Substrates luminol

HRP-based Luminol/H202/ p-iodophenol Oxidized luminol substrate gives off blue light p-iodophenol increases light output Very convenient, sensitive system reaction is detected within a few seconds to 1 hr... 

Recipe is from Schneppenheim et al. (1991). Premixed luminol substrate mix (Mast lmmunosystems, Amersham, Du Pont NEN Renaissance, or Kirkegaard Perry Lumi-GLO) may also be used. For selection of appropriate luminescent solutions, and for definition of abbreviations, see Table B3.4.1. 

C. Detection of probes with luminol substrates (Whitehead et al., 1983 van Gijlswijk... 

J. J.5.2. Luminol substrates for peroxidase Cyclic diacylhydrazides, such as luminol (Fig. 7.11), can be oxidized by POase in the presence of H2O2 (Matthews et al., 1985). Luminol is then converted via an... 

Initially, 50 c illary of 10 fxg mL mbbit anti-mouse IgG in 50 mM sodium carbonate buffer solution, pH 9.6 was incubated overnight at 4 °C. The capillaries were washed three times with PBS-Tl, and then twice with water. Thereafter, 50 pL of 10 pg mL anti-2,4-D IgG in PBS-T2 was added to each capillary, and incubated for 1 h at 37 °C. The capillaries were washed with washing b er and water. Thereafter, 50 pL mixture of 2,4D-TOP and standards or sanqrles were added to each capillary, and the competitive reaction was allowed to proceed by incubation for 2 h at RT. To determine nonspecific binding, four c illaiies received non specific IgG and no pesticide, while ten capillaries received specific IgG but no pesticide, in order to determine the maximum chemiluminescent intensity for the bound fraction. Finally, the capillaries were washed and 50 pL capillary of luminol substrate solution was added, and the maximum chemiluminescent intensity was measured with the modified Hamamatsu PMT. 

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

Chemiluminescent labels, in which the luminescence is generated by a chemical oxidation step, and bioluminescent labels, where the energy for light emission is produced by an enzyme-substrate reaction, are additional labeling types (39,42). Luminol [521 -31 -3] CgHyN202, and acridine [260-94-6] C H N, derivatives are often used as chemiluminescent labels. 

The relationship between CL intensity and time is expressed by a kinetic equation including the reaction rate constants and the substrate concentration. Such is the case with the specific equation for the CL of the luminol reaction, which is one of the most widely studied in this context ... 

The lack of selectivity can be circumvented by coupling a postcolumn flow system to a liquid chromatograph. This has promoted the development of a number of efficient liquid chromatography-CL approaches [16, 17]. Eluted analytes are mixed with streams of the substrate and oxidant (in the presence or absence of a catalyst or inhibitor) and the mixed stream is driven to a planar coiled flow cell [18] or sandwich membrane cell [19] in an assembly similar to those of flow injection-CL systems. Many of these postcolumn flow systems are based on an energy-transfer CL process [20], In others, the analytes are mixtures of metal ions and the luminol-hydrogen peroxide system is used to generate the luminescence [21],... 

Hydrogen peroxide produced as a result of reactions of oxidase enzymes with analyte substrates can be sensitively determined, both directly by luminol ECL and indirectly by Ru(bpy)32+ ECL. For the latter, hydrogen peroxide is detected on the basis of its ability to diminish the ECL reaction between Ru(bpy)32+ and added oxalate, by reacting with, and depleting the concentration of, oxalate. Thus ECL intensity is inversely proportional to the concentration of analyte. This principle has been used, for example, to determine cholesterol [70],... 

Figure 13 Schematic representation of the coupled substrate-enzymatic-luminol CL reaction system in a reversed micellar medium. (From Ref. 64 with permission.)...

Although substituted phenols (e.g., para-iodophenol, para-phenylphenol, firefly luciferin, coumaric acid) are popular enhancers, in both luminol and acridan ester oxidation, enhancers with other functional groups [24], e.g., phe-nylboronic acids [25-28], phenothiazines [29], are also useful. As an example the structure of the phenothiazine enhancer used in the Supersignal substrate family is shown in Figure 6. 

Recently, two major enzyme-catalyzed chemiluminescent reactions have become popular. These use either luminol as a substrate of peroxidase or 3-(2 -spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD) as a substrate of alkaline phosphatase (ALP). 

Luminol semiquinone is further oxidized to luminol endoperoxide, which elicited CL at decomposition. It should be added that in our experiments peroxynitrite-stimulated luminol CL in cells was enhanced in the presence of the NO synthase substrate L-arginine and sharply diminished in the presence of NO synthase inhibitors. Thus, the application of substrates and inhibitors of NO synthases may discriminate luminol-amplified CL stimulated by superoxide and peroxynitrite. 

The enhanced chemiluminescence associated with the autoxidation of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) in the presence of trace amounts of iron(II) is being used extensively for selective determination of Fe(II) under natural conditions (149-152). The specificity of the reaction is that iron(II) induces chemiluminescence with 02, but not with H202, which was utilized as an oxidizing agent in the determination of other trace metals. The oxidation of luminol by 02 is often referred to as an iron(II)-catalyzed process but it is not a catalytic reaction in reality because iron(II) is not involved in a redox cycle, rather it is oxidized to iron(III). In other words, the lower oxidation state metal ion should be regarded as a co-substrate in this system. Nevertheless, the reaction deserves attention because it is one of the few cases where a metal ion significantly affects the autoxidation kinetics of a substrate without actually forming a complex with it. 

In common with all other sensitive detection systems, maintenance of the label enzyme in its active state is important. The precautions detailed in Notes 1—3 should be observed to maximize the sensitivity achieved. Reagents for enhanced chemiluminescence can be prepared in the laboratory or ure available commercially (see Note 4). The purity of the substrate solution is important in achieving maximum sensitivity. Therefore, the precautions detailed in Notes 5-7 should be followed if preparing substrate solutions. The free base form of lummoi undergoes rearrangement ro a mixture of luminol and a series of contaminants. Therefore, luminol should be purified by recrystaliistation as the sodium salt before use (see Note 8). 

Working enhanced chemiluminescence substrate solution (see Note 9)—either a. /Modophenol-enhanced substrate luminol 1.25 mM, p-iodophenol 4 pM,... 

In order to ensure stable concentrations, the anhydrous form of sodium luminol is preferred molecular weight 199 1. This should be stored over silica gel in the dark. Luminol solutions should be stored in the dark at 4°C. Stock solutions must be made up at least weekly and working substrate daily. 

---