An investigation of the in-plane chemically ordered atomic laminates (Mo2/3Sc1/3)2AlC and (Mo2/3Y1/3)2AlC from first principles.

We have calculated electronic, vibrational, and elastic properties of (Mo2/3Sc1/3)2AlC and (Mo2/3Y1/3)2AlC, two recently discovered nanolaminated materials in the family of so-called i-MAX phases. A comparison is made to the properties of the related hypothetical MAX phases Sc2AlC, Y2AlC, and Mo2AlC. From an analysis of the electronic band structures and projected crystal orbital Hamilton populations (pCOHP), we show that the i-MAX phases have more isotropic band structures than the MAX phases, but that their bonding characteristics are very similar, despite belonging to different space groups. However, the similar bonding notwithstanding, qualitative as well as significant quantitative differences are seen in the phonon density of states (PDOS). We also compare the Voigt-Reuss-Hill (VRH) bulk, shear, and Young's moduli. For (Mo2/3Sc1/3)2AlC, BVRH = 132 GPa, GVRH = 89 GPa, and EVRH = 218 GPa, all of which are higher values than for Sc2AlC, but lower than for Mo2AlC. For (Mo2/3Y1/3)2AlC, BVRH = 117 GPa, GVRH = 85 GPa, and EVRH = 205 GPa, which are higher than for Y2AlC, but lower than for Mo2AlC.

Phase stability of the nanolaminates V2Ga2C and (Mo1-xVx)2Ga2C from first-principles calculations.

We here use first-principles calculations to investigate the phase stability of the hypothetical laminated material V2Ga2C and the related alloy (Mo1-xVx)2Ga2C, the latter for a potential parent material for synthesis of (Mo1-xVx)2C, a new two-dimensional material in the family of so called MXenes. We predict that V2Ga2C is thermodynamically stable with respect to all identified competing phases in the ternary V-Ga-C phase diagram. We further calculate the stability of ordered and disordered configurations of Mo and V in (Mo1-xVx)2Ga2C and predict that ordered (Mo1-xVx)2Ga2C for x≤ 0.25 is stable, with an order-disorder transition temperature of ∼1000 K. Furthermore, (Mo1-xVx)2Ga2C for x = 0.5 and x≥ 0.75 is suggested to be stable, but only for disordered Mo-V configurations, and only at elevated temperatures. We have also investigated the electronic and elastic properties of V2Ga2C; the calculated bulk, shear, and Young's modulus are 141, 94, and 230 GPa, respectively.

Extraction and determination of adenosine 5'-triphosphate in bovine milk by the firefly luciferase assay.

The release of ATP from somatic cells in milk with the detergent Triton X-100 was optimized for assay with firefly luciferase. A small volume of milk (40 microliters) is added to 0.8 ml of 0.2% Triton X-100 in 100 mM Tris, 4 mm EDTA, pH 7.8. After approximately 1 min, 0.2 ml of luciferase reagent is added and the emission of light is measured in a luminometer. Results are calibrated with an ATP standard. This single method gave high yields of ATP from somatic cells in milk without interference from bacterial ATP. Extracts could be stored or transported prior to assay without deterioration of results. A close correlation was found between somatic cell count and ATP in milk samples collected at a farm as well as in milk samples from a cow with experimental mastitis. Results are promising for future use for diagnosis of mastitis but further work and field testing has to be done before it can be used on a wider scale.

Chemiluminescent immunosorbent assay of serum myoglobin based on the luminol reaction.

A simple and sensitive method for the determination of serum myoglobin is described. Myoglobin was determined in the range of 10-500 micrograms/1 by the chemiluminescent luminol reaction after adsorption to anti-myoglobin IgG onto a solid phase. Only 50 microliter of serum has to be used and the luminescent immunoassay (LIA) has a better sensitivity and a wider linear range than the conventional radioimmunoassay (RIA). However, in its present form LIA has a higher imprecision than RIA. In clinical specimens the correlation between the two methods was excellent.

Methodological aspects on the firefly luciferase assay of adenine nucleotides in whole blood and red blood cells.

The firefly luciferase assay of adenosine-5'-triphosphate (ATP) is used in a simple procedure for the determination of adenine nucleotides in whole blood and red blood cells. Cells are lysed with cold trichloroacetic acid and nucleotides are determined in the lysate after dilution with buffer. In a comparative study with the spectrophotometric assay of ATP, extracts were prepared by centrifugation of the lysate to remove interfering turbidity. This step resulted in a significant loss of ATP in the clear supernatant. This loss could be avoided by determination of ATP by the firefly luciferase assay in diluted uncentrifuged lysates. The yields of ATP, ATP + ADP and ATP + ADP + AMP were compared in a red blood cell preparation after lysis with trichloroacetic acid and another lytic method based on the detergent triton X-100. The precision was better with acid whereas yields were essentially the same with both methods. The method of standardization, external or internal, was of little practical importance regarding yields and precision, but internal calibration is recommended to ensure that there is no interaction of the luciferase reaction with lytic reagent or sample.

Leakage of adenylate kinase from stored blood cells.

The bioluminescent firefly luciferase assay for ATP was used to measure adenylate kinase activity in plasma. The formation of ATP from ADP was measured continuously in a coupled assay using a luminometer. Optimal analytical conditions were determined for the coupled reaction. The assay was used to follow accumulation of adenylate kinase in plasma of different preparations of stored red blood cells. Adenylate kinase was found to be released concomitantly with hemoglobin during aging. There was a high degree of correlation between the amount of accumulated hemoglobin and adenylate kinase. The assay was also used to measure lysis of stored platelets during aging.

Bioluminescent assay for total bile acids in serum with use of bacterial luciferase.

This simple, rapid, sensitive kinetic bioluminescent method for the assay of bile acids in serum involves use of 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) in combination with a new, commercially available NADH Monitoring Reagent (LKB-Wallac) containing a low activity of NADH:FMN-oxidoreductase and a high activity of bacterial luciferase. Interfering dehydrogenases in serum are inactivated in the test tube with trichloroacetic acid before the assay. The standard curve is linear for concentrations of bile acids up to about 300 mumol/L. With a sample volume of 20 microL, the detection limit is about 0.2 mumol/L. The within-run precision (CV) is about 10%, both at high and low concentrations of bile acids in serum. Correlation is good (r = 0.996) between results by this method and an enzymatic method based on spectrophotometry. However, the latter method is considerably less sensitive, and it is less precise at low concentrations of bile acids.

Firefly luciferase ATP assay as a screening method for bacteriuria.

A rapid (15 min) test for bacteriuria based on firefly luciferase analysis of bacterial ATP has been evaluated in 2,018 clinical urine specimens. The test procedure involves removal of nonbacterial ATP by treatment of urine with Triton X-100 and apyrase, extraction of bacterial ATP by boiling, and bioluminescent analysis of bacterial ATP by firefly luciferase, using a luminometer. For comparison, the widely used nitrite test was included in the study as an example of an alternative rapid chemical test. The test was set up to distinguish between specimens yielding greater than 10(5) CFU/ml and specimens yielding less than 10(5) CFU/ml. A level of 13.5 nM ATP was chosen to define the limit between negative and positive results. At this discriminatory level, 92% of specimens yielding greater than 10(5) CFU/ml and 88% of specimens yielding less than 10(5) CFU/ml were correctly classified with the luciferase method, whereas corresponding figures for the nitrite test were 55 and 99%, respectively. Of the 12% false luciferase positives, 20% were shown to contain greater than 10(5) CFU/ml on prolonged incubation, thus reducing the false-positive rate to 10%. Of the 8% false luciferase negatives, 65% had low levels of CFU in the range of 10(5) to 10(6).

Preservation of red blood cells: studies of erythrocyte adenine nucleotides using luminescence analysis.

The levels of ATP and total adenine nucleotides (ATP + ADP + AMP) were determined by firefly luciferase assay in red blood cells during storage for 5 weeks at 4 degrees C. With few exceptions, no significant differences in nucleotide levels were found between whole blood stored in CPD-adenine and various preparations of red blood cells in CPD-adenine or CPD with saline-adenine-glucose (SAG) as additive. The levels of ATP and total adenine nucleotides during storage are discussed in relation to glucose levels, extracellular pH and shelf life of the red blood cells.

A simple, fully enzymic bioluminescent assay for triglycerides in serum.

In this fully enzymic bioluminescent assay, triglycerides are cleaved by lipase (EC 3.1.1.3) and carboxylesterase (EC 3.1.1.1), and the glycerol obtained is phosphorylated with ATP and glycerol kinase (EC 2.7.1.30). The ATP consumed in the latter reaction is determined by the firefly luciferin-luciferase reaction, and corresponds to the concentration of triglycerides. All the enzymic reactions and the bioluminescent reading can be performed in the same test tube. The precision (CV) of the assay varied between 1 and 7% at different concentrations with a fully enzymic method based on spectrophotometry (r = 0.98).

A sensitive method for determination of serum hemoglobin based on iso-luminol chemiluminescence.

A simple and rapid method for determination of serum hemoglobin is described. Hemoglobin may be determined in serum within the range of 0.02-400 mg/1 by the sensitive chemiluminescent iso-luminol reaction. The iso-luminol assay was considerably more sensitive than the conventional colorimetric procedure based on tetramethylbenzidine. Precision and accuracy were higher with the iso-luminol assay especially at low levels of hemoglobin. The correlation between the luminescent and colorimetric method was linear but the colorimetric determinations resulted in higher concentrations of hemoglobin. This discrepancy was probably caused by non-heme serum iron which interfered more strongly with the colorimetric method.

The relationship between luminol chemiluminescence and killing of staphylococcus aureus by neutrophil granulocytes.

Luminol chemiluminescence induced by phagocytosis of bacteria was studied in a system consisting of polymorphonuclear granulocytes (PMN), serum, luminol and Staphylococcus aureus. To evaluate the quantitative relationship between luminol chemiluminescence and the bactericidal process time courses for both variables were compared. It was found that initial rate of increase of chemiluminescence and initial rate of killing of bacteria were well correlated whereas the correlation was poorer for later stages of the process. When the rate of the bactericidal process was varied by changing concentrations of bacteria and PMN, directly proportional variations of initial rates of increase of chemiluminescence were observed. This is interpreted as reflecting an accumulation of oxidizing radicals as the result of a phagocytosis dependent gradual activation of the NADPH oxidase system, leading to luminol oxidation and/or killing of bacteria. However, by thermal inactivation of PMNs, chemiluminescence could be diminished whereas killing remained essentially unaffected, showing that these two processes could be uncoupled. Also, addition of erythrocytes to the PMN suspension was associated with decreased chemiluminescence and lysis of erythrocytes with an increased chemiluminescence, emphasizing the importance of proper control of the components of the leucocyte test suspension.

Sensitive kinetic bioluminescent assay of glycerol release from human fat cells.

A sensitive and accurate assay of lipolysis has been developed, measuring the rate of glycerol release from fat cells with a bioluminescent assay. The rate of glycerol-dependent ATP-consumption in a system consisting of glycerokinase, ATP, luciferin, and luciferase was determined kinetically as a decrease of the ATP-induced luminescence and used for calculation of the concentration of glycerol. Under the conditions employed, it was possible to measure the concentration of glycerol down to a level of about 0.5 micron mol/l. Under the same conditions, the detection limit of the usual fluorometric method was about 15 micron mol/l. The coefficient of variation obtained with the bioluminescent assay was 11% at a level of 1.0 micron mol of glycerol, 2-6% at a level of about 5 micron mol/l, and 1-3% at a level of about 20 micron mol/l. Satisfactory results were obtained in different recovery experiments. Using human fat cells, it was possible to determine the rate of glycerol release with a cell concentration in the medium of only 5,000-10,000 cells/ml. It is concluded that the bioluminescent assay of glycerol release should be preferred when there is a demand for sensitivity, e.g., when the rate of lipolysis is low and when only a small amount of biopsy material is available.

Luminescence immunoassay (LIA): a solid-phase immunoassay monitored by chemiluminescence.

A luminescence immunoassay (LIA) has been developed utilizing the chemiluminescent luminol reaction with heme as catalyst. Rabbit antibody against human serum albumin was quantitated in antigen coated plastic tubes using commercially available goat anti-rabbit IgG conjugated to horseradish peroxidase which was the source of heme. The measurable range of antibody is considerably wider by LIA than by enzyme immunoassays. The time to develop and measure activity is short and constant which makes LIA suitable for automation. In its present form, LIA is slightly less sensitive but has better day-to-day reproducibility than corresponding enzymes immunoassays.

Correlation between hemin content and the chemiluminescent luminol reaction with bacteria.

Protohemin and covalently bound hemin were determined in eight aerobic bacterial strains. A good correlation between protohemin content and luminol reactivity was found. The ratio of luminol reaction to protohemin for the eight investigated strains was essentially identical to that of pure protohemin, 0.7 X 10(16) mV/mol. Covalently bound hemin contributed to the chemiluminescence to a minor extent only (0.7 X 10(14) mV/mol, in accordance with earlier observations of the lower reactivity of cytochrome c and related compounds. A difference in reaction kinetics of the luminol reaction with covalently bound hemin (slower reaction than protohemin) and protohemin was observed in vivo as well as in vitro. The phenomenon could be used to differentiate between strains with different hemin composition.

Effects of ampicillin on intracellular levels of adenosine triphosphate in bacterial cultures related to antibiotic susceptibility.

Short time effects of ampicillin on viability and levels of intracellular ATP were studied in bacterial cultures and a close relationship between intracellular ATP levels and viability was demonstrated. The connection between the effects observed and MIC values is discussed. The possibility of using the phenomenon for rapid antibioitc susceptibility testing was studied in clinical isolates incubated for 2 hours followed by luciferase assay of intracellular ATP. A positive correlation was demonstrated between ampicillin-induced decreases in intracellular ATP and inhibitory zone diameters, as measured by the agar diffusion technique.

A rapid semiautomated bioassay of gentamicin based on luciferase assay of bacterial adenosine triphosphate.

A rapid semiautomated bioassay of gentamicin in serum based on the effect of gentamicin on intracellular adenosine triphosphate (ATP) levels in bacterial cultures is presented. The ATP assay was performed with the firefly luciferin/luciferase system and results were compared with an agar diffusion technique. The lower limit of detection was set at 1 microgram/ml. The accuracy of the ATP assay expressed as a recovery of gentamicin over the range 1--8 microgram/ml was 98--103% and S.D. varied between 11-15%. The reproducibility was approximately within the same limits. Other antibiotics tested did not interfere with the determination of gentamicin. 112 clinical serum specimens containing gentamicin alone (N = 55) or in combination with other antibiotics (N = 57) were assayed and results with the two methods compared (r = 0.94). Volumes required were 0.1 ml serum and results were available within 3 h.