Chemiluminescence background

Figure 15.15 A) Model BSA-biotin, HRP-streptavidin chemiluminescent assay scheme. B) Acridan chemiluminescence emission as a function of time from HRP modified ass coverslips coated with 1 mM BSA-biotin and I mM HRP-streptavidin and positioned glass substrate geometries with and without 12.3 cm Al triangle 7S nm thick (left). C) Acridan chemiluminescence background emission as a function of time for glass coverslips incubated with 1.5% BSA and 1 mM HRP-streptavidin (control) positioned on glass substrate geometries with and without 12.3 cm Al triangle 75 nm thick shapes (right). All samples were ejqiosed to four 10 second microwave pulses (Mw pulse) at 10% power. Adapted from Anal Chem 79 7042-7052 (2007).

The target for optimization in FTA with CL detection is to adjust all experimental factors in such a way so that the detector views as much radiation as possible while the chemiluminescent solution flows through the cell. Hence the kinetics of the flow and detector system should be monitored to match the kinetics of the reaction and generate maximum intensity inside the cell. The effect of experimental variables on the CL signal cannot be exactly predicted in advance and there is not enough theoretical background to support any suggestion. 

A background emission underlies all chemiluminescence spectra obtained in reactions of O atoms. This emission results from the recombination of O atoms,... 

Emissions from five different excited electronic states of 02 throughout the visible spectral region of 400-800 nm have been identified [17], placing a practical limit on the concentration of O atoms that can be used as a chemiluminescence reagent since the background emission increases quadratically in O atom concentration. 

Figure 7 Kinetics of the chemiluminescent HRP-catalyzed peroxidation of GZ-11. The course of the light output, corrected for background (S-B), is plotted versus time for different amounts of HRP (0.1-100 pg).

Figure 8 HRP calibration curve (log-log plot of HRP dose vs. chemiluminescence signal, corrected for background) using signal reagent incorporating GZ-11.

This protocol permits detection of 10 pmol of target DNA dotted on a nylon membrane after hybridization with the d(G)30 probe [15]. The background chemiluminescence caused by nonspecific binding of the probe in the hybridization buffer to the membrane is negligible in this assay system. However, both the target DNA and the cDNA probe bound to the membrane are detected solely... 

Lueking et al. (1999) arrayed recombinant proteins on NC membranes and screened them with different antibodies. Joos and coworkers (2000) printed down autoantigens onto NC membranes and compared performance relative to silylated (aldehyde) and PLL glass slides. Protein arrays could be stored at room temperature for a month without significant loss in activity. Huang (2001) hand spotted down IgC species and antibodies directed toward various cytokines onto membranes. The properties of various commercial membranes were assessed in terms of absorption, background, and sensitivity levels based upon detection by enhanced chemiluminescence (ECL). 

Carbon-14 content is measured by specially designed gas proportional counters (7. Aerosol samples are first converted to CO2 by combustion in a macroscale version of the thermal evolution technique. A clam shell oven was used to heat the sample for sequential evolution of organic and elemental carbon under equivalent conditions. Due to the possibility of thermal gradients, conditions in the macroscale apparatus were adjusted to produce the same recoveries of total carbon (yg C per cm of filter area) as for the microscale apparatus. Carbon-14 data are reported as % contemporary carbon based on the 1978 1 C02 content in the atmosphere. Aldehyde data referred to in this paper were obtained by impinger sampling in dinitrophenylhydrazine/acetonitrile solution and analysis of the derivatives by HPLC with UV detection (12). Olefin measurements were made by a specially designed ozone-chemiluminescence apparatus (13) difficulties in calibration accuracy and background drift with temperature limit its use to inferences of relative reactive hydrocarbon levels. 

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