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Drexler D Barlow DJ Falk P Cantone J Hernandez D Ranasinghe A Sanders M Warrack B McPhee F 《Analytical and bioanalytical chemistry》2006,384(5):1145-1154
Fluorescence detection has been a method of choice in industry for screening assays, including identification of enzyme inhibitors,
owing to its high-throughput capabilities, excellent reproducibility, and sensitivity. Occasionally, inhibitors are identified
that challenge the fluorescence assay limit, necessitating the development of more sensitive detection methods to assess these
compounds. For data mining purposes, however, original assay conditions may be required. A direct method transfer to highly
sensitive and specific LC-MS-based methods has not always been possible due to the presence of MS-incompatible neutral detergents
and non-volatile salts in the assay matrix. Utilizing an in vitro proteolytic screening assay for the serine protease hepatitis C virus (HCV) nonstructural (NS) 3 protease as a test case,
we report the development of an automated sample clean-up procedure implemented on-line with liquid chromatography–tandem
mass spectrometry (LC-MS/MS) analysis to complement fluorescence detection. Ion exchange and peptide microtraps were employed
to remove MS-incompatible assay matrix components. Three protease inhibitors were used to validate the MS/MS method. Comparable
potencies were achieved for these compounds when assessed by fluorescence and MS/MS detection. Furthermore, four-fold less
enzyme could be utilized when employing the MS/MS method compared to fluorescence detection. The longer analysis time, however,
resulted in reduced sample capacity. The potency of our designed HCV NS3 protease inhibitors are thus routinely evaluated
using a continuous fluorescence-based assay. Only pertinent inhibitors approaching the fluorescence assay sensitivity limit
are subsequently analyzed further by LC-MS/MS. This methodology allows us to maintain a database and to compare results independent
of the detection method. Despite the relatively slow sample turnaround time of this LC-MS approach, the versatility of the
automated on-line clean-up procedure and sample analysis can be applied to assays containing reagents which were historically
considered to be MS incompatible. 相似文献
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冷原子的双阱微磁表面囚禁 总被引:2,自引:0,他引:2
提出了两种新颖的采用载流导线的双阱微磁表面囚禁方案(即双U形与双Z形导线囚禁)。通过改变囚禁方案中直导线中的电流方向,即可将双U形导线囚禁改变为双Z形导线囚禁;如果逐渐减小直导线中的电流大小,即可将一个双阱微磁囚禁连续地合并为一个单阱微磁囚禁,反之亦然。详细计算和分析了上述两种载流导线囚禁方案的磁场及其梯度的空间分布。研究发现在导线中通以较小的电流,即可在导线表面附近产生很大的磁场梯度及其曲率。例如当电流为O.2A时,其磁场梯度和曲率可分别达到0.2T/cm和10T/cm2以上。由于双U形导线囚禁中存在磁场零点,而双Z形导线囚禁中仅存在磁场最小值,所以双U形导线囚禁仅适用于制备双样品磁光囚禁(MOT)或研究中性原子的冷碰撞,而双Z形导线囚禁除了可用于研究原子的冷碰撞之外,还可以用于制备双样品玻色-爱因斯坦凝聚(BEC)或实验研究双阱玻色-爱因斯坦凝聚的性质等。 相似文献
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