PubMed · 13205113
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J SONKA, V ZBUZEK, J V KOSTIR. 1954. [Nonspecific Ehrlich's reaction].. https://pubmed.ncbi.nlm.nih.gov/13205113/
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Magnetron co-sputtering was used to produce silicon-doped hydroxyapatite (Si-HA) as coatings intended for potential applications such as orthopedic and dental implants. It was found that the crystallinity of the as-sputtered coatings increased after annealing, resulting in a nanocrystalline apatite structure. Subsequently, the bioactivity of the coatings was evaluated in an acellular simulated body fluid (SBF). Physicochemical evaluation demonstrated that a carbonate-containing apatite layer, which is essential for bonding at the bone/implant interface, was formed on the coating surfaces after immersion in SBF between 4 and 7 days. The annealed coatings exhibited enhanced bioactivity and chemical stability under physiological conditions, as compared with the as-sputtered coatings. It is proposed that the rate at which the carbonate-containing apatite layer forms is dependent on the scale factor of the structure. A nanocrystalline structure can provide a higher number of nucleation sites for the formation of apatite crystallites, leading to a more rapid precipitation of carbonate-containing apatite layer. This work shows that Si-HA coatings offer considerable potential for applications in hard tissue replacement, owing to their ability to form a carbonate-containing apatite layer rapidly.
A simple experimental approach for studying and identifying the relative matrix effect (for example "plasma-to-plasma" and/or "urine-to-urine") in quantitative analyses by HPLC-MS/MS is described. Using as a database a large number of examples of methods developed in recent years in our laboratories, the relationship between the precision of standard line slopes constructed in five different lots of a biofluid (for example plasma) and the reliability of determination of concentration of an analyte in a particular plasma lot (or subject) was examined. In addition, the precision of standard line slopes was compared when stable isotope-labeled analytes versus analogs were used as internal standards (IS). Also, in some cases, a direct comparison of standard line slopes was made when different HPLC-MS interfaces (APCI versus ESI) were used for the assay of the same compound, using the same IS and the same sample preparation and chromatographic separation conditions. In selected cases, the precision of standard line slopes in five different lots of a biofluid was compared with precision values determined five times in a single lot. The results of these studies indicated that the variability of standard line slopes in different lots of a biofluid [precision of standard line slopes expressed as coefficient of variation, CV (%)] may serve as a good indicator of a relative matrix effect and, it is suggested, this precision value should not exceed 3-4% for the method to be considered reliable and free from the relative matrix effect liability. Based on the results presented, in order to assess the relative matrix effect in bioanalytical methods, it is recommended to perform assay precision and accuracy determination in five different lots of a biofluid, instead of repeat (n=5) analysis in the same, single biofluid lot, calculate standard line slopes and precision of these slopes, and to use <3-4% slope precision value as a guide for method applicability to support clinical studies. It was also demonstrated that when stable isotope-labeled analytes were used as internal standards, the precision of standard line slopes in five different lots of a biofluid was </=2.4% irrespective of the HPLC-MS interface utilized. This clearly indicated that, in all cases studied, the use of stable isotope-labeled IS eliminated relative matrix effect. Also, the utilization of the APCI interface instead of ESI led to the elimination of the relative matrix effect in all cases studied. When the precision of standard line slope values exceeds the 3-4% limit, the method may require improvements (a more efficient chromatography, a more selective extraction, a stable isotope-labeled IS instead of an analog as an IS, and/or a change in the HPLC-MS interface) to eliminate the relative matrix effect and to improve assay selectivity.
Quantitative protein bioanalysis in complex biological fluids presents considerable challenges in biological studies and disease diagnosis. The major obstacles are the background signals from both the probe and the biological fluids where the proteins reside. We have molecularly engineered light-switching excimer aptamer probes for rapid and sensitive detection of a biomarker protein, platelet-derived growth factor (PDGF). Labeled with one pyrene at each end, the aptamer switches its fluorescence emission from approximately 400 nm (pyrene monomer) to 485 nm (pyrene excimer) upon PDGF binding. This fluorescence wavelength change from monomer to excimer emission is a result of aptamer conformation rearrangement induced by target binding. The excimer probe is able to effectively detect picomolar PDGF in homogeneous solutions. Because the excimer has a much longer fluorescence lifetime (approximately 40 ns) than that of the background (approximately 5 ns), time-resolved measurements were used to eliminate the biological background. We thus were able to detect PDGF in a cell sample quantitatively without any sample pretreatment. This molecular engineering strategy can be used to develop other aptamer probes for protein monitoring. Combined with lifetime-based measurements and molecular engineering, light-switching excimer aptamer probes hold great potential in protein analysis for biomedical studies.