第三章多标记仪讲述.ppt

Description of the slide contents Sensitivity of conventional fluorometric assay is very much background limited, during the fluorometric analysis there are interfering background sources, such as sample and reagent fluorescence, plastics and optics luminescence and detector noise, which greatly adversely affect the sensitivity of the analysis. By applying temporal resolution, i.e using a pulsed excitation and measuring the fluorescence intensity after a certain delay time has elapsed, you can avoid the interference created by short living background (sample and reagent fluorescence decay time 2-10 ns, plastics 0-50 μs, optics 0-1000 μs, detector noise is constant). Wallac has adopted a millisecond TR systems (time domain) where the specific signal of long decay (millisecond duration) is simply but completely distinguished from the background noise of short decay. Message Wallac TR at millisecond time frame gives a vary efficient temporal discrimination = robust and sensitive detection but = requires a label of millisecond decay time = possible only with lanthanide chelates Note! FLARE is μs TR applying frequence domain and not time domain, and does not achieve a similar sensitivity * ATP Determinations: All living cells contain ATP. ATP can be extracted from cells and assayed with firefly luciferase. In this luminescent reaction, ATP is the limiting reagent. Therefore, the light reaching the luminometers photomultiplier tube is proportional to the amount of ATP in the sample and, correspondingly, to the number of cells from which it was extracted. ATP luminometric assays have been used for decades. ATP measurements are used in: Microbial Contamination of Water, Foods Pharmaceuticals: ? Biomass Estimations: Microbiologists use ATP determinations to measure total live biomass in soil and sediments. Related studies include determining the effect of pollutants on fresh water and marine micro-organisms. ? Clinical Research: Clinical research applications inc