Liquid Chromatography with Fluorescence Detection in the Analysis of Biological FluidsRoth, Marc; Uebelhart, Daniel
doi: 10.1080/00032710008543195pmid: N/A
Liquid chromatography with fluorescence detection is well suited to the analysis of biological fluids, as it combines both selectivity and sensitivity. The determinations are not limited to fluorescent compounds, as non-fluorescent substances can be converted to fluorescent derivatives by appropriate reactions. As a consequence of progress in methodology and of the development of new reagents, a great number of biological substances and drugs can now be successfully analyzed by this technique. Reliable automated procedures using pre-column derivatization are available, in particular for the analysis of amino acids and amines. In addition, systems using short columns, reduced particle size of the stationary phase and ultramicro detector cells represent a promising approach to the analysis of very small volumes of sample.
Amperometric Glucose Biosensor Based on the Deposition of Copper and Glucose Oxidase onto Glassy Carbon TransducerRodriguez, M. C.; Rivas, G. A.
doi: 10.1080/00032710008543196pmid: N/A
The performance of a first generation glucose amperometric biosensor based on the entrapment of glucose oxidase (GOx) within a net of copper electrodeposited onto activated glassy carbon electrode, is described. The copper electrodeposited offers an efficient electrocatalytic activity towards the reduction of enzymatically-liberated hydrogen peroxide, allowing for a fast and sensitive glucose quantification. The influence of the electrodeposition conditions (pH, potential, time, copper salt and enzyme concentrations) on the response of the bioelectrode was evaluated from the amperometric signals of hydrogen peroxide and glucose. The combination of copper electrodeposition with a nation membrane allows an excellent selectivity towards easily oxidizable compounds such as uric and ascorbic acids at an operating potential of -0.050 V. The response is linear up to 2.0 × 10−2 M glucose, the detection limit being 1.2 × 10−3 M.
Quantitation of Monosodium Glutamate Using Immobilized Glutamate Oxidase/Peroxidase and Flow Injection AnalysisMankasingh, U.; Narinesingh, D.; Ngo, T.T.
doi: 10.1080/00032710008543198pmid: N/A
A rapid and sensitive flow injection method is developed for the quantitation of glutamate in food samples. The method incorporates a covalently immobilized glutamate oxidase and peroxidase bioreactor linked in tandem. The H2O2 liberated from the glutamate samples as a result of enzymatic action is monitored spectrophotometrically at 552 nm using 4-aminoantipyrine and 3-dimethylaminobenzoic acid as a new chromogenic reagent. Glutamate calibration curves are linear up to 140 μM with a detection limit of 1 μM. Recovery yields from soup matrices are in the range 97 – 101%. Inter- and intra-day precision studies gave CV's of less than 3.5%. The immobilized enzymes show good storage and operational stabilities. Up to 40 samples h−1 can be manually analyzed. Excellent correlation is obtained from a comparison of the glutamate content of various soup brands and matrix type (solid, condensed and broths) obtained by the proposed FI method with those obtained for the same samples analyzed by the standard reference method (y 0.99x + 0.034).
Studies on Electrocatalytically Kinetic Behavior of Horseradish Peroxidase at Salt Bridge Supported Bilayer Lipid MembraneZhang, Yan-Li; Zhang, Chun-Xu; Shen, Han-Xi
doi: 10.1080/00032710008543199pmid: N/A
This paper reports a novel and biological compatible biosensor that was used to study the electrochemical behavior of horseradish peroxidase (HRP) incorporated in salt bridge-supported bilayer lipid membrane (Sb-BLM). The incorporation of HRP was achieved by electrostatic interaction between HRP and LA anion sites pre-incorporated in Sb-BLM, which result in the enhancement of the electron exchange between the protein and the electrode surface. A quasi-reversible electron transfer of HRP was observed even in the absence of mediators. The electrocatalytically kinetic behavior of HRP and the electrode-kinetic process were investigated with this biosensor. In the presence of hydrogen peroxide, the electrocatalytical activity of HRP was determined.
Triglyceride Assay by Amperometric Microbial Biosensor: Sample Hydrolysis and Kinetic ApproachTkáč, Ján; Švitel, Juraj; Novák, Richard; Šturdik, Ernest
doi: 10.1080/00032710008543200pmid: N/A
A triglyceride assay based on triglyceride hydrolysis and glycerol detection was developed. Non-specific lipase isolated from Candida rugosa and intact Gluconobacter oxydans cells, containing membrane-bound glycerol dehydrogenase, were used to develop a biosensor. Two approaches were investigated: analysis of pre-hydrolysed samples and a kinetic approach. The sensor prepared from G. oxydans cells exhibited sensitive and fast response to glycerol: detection limit 20 μM (S/N=3), linear range up to 2 mM and response time 84 s (90% of steady-state). The triglyceride assay of pre-hydrolysed samples was based on a 20 min hydrolysis and determination of released glycerol by the biosensor. A calibration curve linear up to 12 mM was obtained for triolein samples. The kinetic approach was based on simultaneous glyceride hydrolysis and glycerol detection. Analysis time of 10 min, linear range up to 30 mM, and estimated detection limit of 50 μM were achieved using the kinetic approach. The kinetic triglyceride assay is not influenced by free glycerol present in a sample. Storage stability, expressed as a half life (50% of the initial response), was 7 days when trehalose was used as a stabiliser.
Characterization of the Binding of Methylene Blue to DNA by Spectroscopic MethodsLi, Wen-You; Xu, Jin-Gou; He, Xi-Wen
doi: 10.1080/00032710008543201pmid: N/A
Methylene blue (MB) binds to the double helical DNA with a high affinity, as deduced from the absorption and fluorescence spectral data. Extensive hypochromism and red shifts in the absorption spectra were observed when MB binds to calf thymus DNA(CT DNA), which suggested the intercalation mechanism of MB into DNA bases. Upon binding to DNA, the fluorescence from MB was efficiently quenched by the DNA bases, with no shifts in the emission maximum. The large increases in the polarization upon binding to CT DNA supported the intercalation of MB into the helix. Ferrocyanide quenching studies showed that the magnitude of Ksv of the bound MB was lower than that of the free MB. The results of competitive binding studies showed that ethidium bromide could be displaced by MB from ethidium-DNA complex. The results of all above further studies also proved the intercalation of MB into DNA base stack.
Simultaneous Quantification of Ceftazidime and Pyridine, its Main Degradation Product, by High-Performance Liquid ChromatographyFavetta, P.; Janoly, A.; Allombert, C.; Breysse, C.; Guitton, J.; Bureau, J.
doi: 10.1080/00032710008543202pmid: N/A
A high performance liquid chromatographic method for the simultaneous assay of ceftazidime and pyridine, the principal degradation product of ceftazidime, is described. The method was fully validated in terms of recovery, linearity, selectivity and precision. An application was done on stability of ceftazidime at 40 mg.mL1 in infusion solutions 0.9% NaCl and 5% glucose on 24 hours in ambulatory infusion device. Quantification results of pyridine were more linear and accurate than ceftazidime. Pyridine was a good label in ceftazidime stability studies.