Chemiluminescence peroxide assay

Mansouri A, Makris DP and Kefalas P. 2005. Determination of hydrogen peroxide scavenging activity of cinnamic and benzoic acids employing a highly sensitive peroxyoxalate chemiluminescence-based assay structure-activity relationships. J Pharm Biomed Anal 39(l-2) 22-26. 

Red wines have been shown to have higher antioxidant capacity than rose and white wines when analyzed by a linoleic acid peroxidation assay based on measuring chemiluminescence (Kondo et al., 1999). The higher antioxidant activity was related... 

The antioxidant activity of alizarin was established in four different assays (1) suppression of light emission in the p-iodophenol enhanced chemiluminescent assay, (2) scavenging of superoxide anion (02 -) in a hypoxanthine-xanthine oxidase system, (3) protection of rat liver microsomes from lipid peroxidation by ADP/iron(II) ions, and (4) protection of bromobenzene-intoxicated mice from liver injury in vivo [141]. Alizarin was compared with Trolox (water soluble vitamin E), the flavonoid baicalin and green tea proanthocyanidins. In assay (1) the activity of alizarin was 76% of that of Trolox. In assay (2) the inhibition of 02 -induced chemiluminescence was 40%, 32%, 23% and 14% for Trolox, alizarin, green tea polyphenols and baicalin respectively. Alizarin was not significantly active in the lipid peroxidation assay but after baicalin the most active compound in the in vivo assay. This shows again the difficulty in the evaluation of antioxidant activity and the differences between in vitro and in vivo assays [141]. 

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). 

A method of detecting herbicides is proposed the photosynthetic herbicides act by binding to Photosystem II (PS II), a multiunit chlorophyll-protein complex which plays a vital role in photosynthesis. The inhibition of PS II causes a reduced photoinduced production of hydrogen peroxide, which can be measured by a chemiluminescence reaction with luminol and the enzyme horseradish peroxidase (HRP). The sensing device proposed combines the production and detection of hydrogen peroxide in a single flow assay by combining all the individual steps in a compact, portable device that utilises micro-fluidic components. 

Applications of the oxalate-hydrogen peroxide chemiluminescence-based and fluorescence-based assays with NDA/CN derivatives to the analysis of amino acids and peptides are included. The sensitivity of the chemiluminescence and fluorescence methods is compared for several analytes. In general, peroxyoxalate chemiluminescence-based methods are 10 to 100 times more sensitive than their fluorescence-based counterparts. The chief limitation of chemiluminescence is that chemical excitation of the fluorophore apparently depends on its structure and oxidation potential. 

Bioluminescence and chemiluminescence are very powerful analytical tools, since in addition to the direct measurement of ATP, NAD(P)H or hydrogen peroxide, any compound or enzyme involved in a reaction that generates or consumes these metabolites can be theoretically assayed by one of the appropriate light-emitting reactions. Some of these possibilities have been exploited for the development of optical fibre sensors, mainly with bacterial bioluminescence and with luminol chemiluminescence. 

Figure 3.29.A shows a flow-cell of 20 iL inner volume used to hold immobilized anti-mouse IgG bound to a rigid beaded support (activated Pierce trisacryl GF-2000). The cell was used to develop a two-site immunoassay for mouse IgG by consecutive injection of the sample, acridinium ester-labelled antibody and alkaline hydrogen peroxide to initiate the chemiluminescence, which started the reaction sequence shown in Fig. 3.29.B. Regenerating the sensor entailed subsequent injection of an acid solution, which resulted in a determination time of ca. 12 min (this varied as a fimction of the flow-rate used, which also determined the detection limit achieved, viz. 50 amol for an overall analysis time of 18 min) [218]. The sensor was used for at least one week with an inter-assay RSD of 5.9%. Attempts at automating the hydrodynamic system for use in routine analyses are currently under way.

The LIA is an immunoassay in which the antigen or antibody are labeled with either a chemiluminescent or bioluminescent tags (41, 58). Luminescent molecules are produced by oxidation reactions. Bis-phenyl oxalates in presence of hydrogen peroxides are used for chemiluminescent assays and luciferin in presence of luciferase enzyme is used for bioluminescent assays. The sensitivity of the LIA s are in the pg/ml or lower range. 

Several other chromophores have been used in the development of sensors based upon ECL. For example, the luminol reaction is a conventional chemi-luminence reaction that has been studied in detail and it is believed that the mechanism of the ECL reaction is similar, if not identical, to that of the chemiluminescence. As shown in Fig. 2, the luminol ion undergoes a one-electron oxidation to yield a diazaquinone, which then reacts with peroxide or superoxide ( OOH) to give the excited 3-aminophthalate which has an emission maximum of 425 nm. This reaction is particularly versatile and has been utilized in a variety of ECL assays, many of which have been previously summarized by Knight [1], The luminol ECL reaction can be used for the determination of any species labeled with luminol derivatives, hydrogen peroxide, and other peroxides or enzymatic reactions that produce peroxides. A couple of examples are described later. 

Chemiluminescence of oxidized luminol has been the basis of several lumino-metric methods of estimation of TAC (Table 1). The mostcommon is to measure the induction time of the reaction. Often the chemiluminescence is first induced by an oxidant and then attenuated by addition of a sample, and the time to recover the initial fluorescence is measured. The enhanced chemiluminescent assay introduced a decade ago is based on the oxidation of luminol by perborate or by hydrogen peroxide in a reaction catalyzed by horseradish peroxidase. Enhancement (and stabilization) of luminescence is achieved by addition of p-iodophenol. The original procedure used a commercial reagent kit (ECL Anti-oxidant Detection Pack... 

The immediate appearance of lipid hydroperoxides after irradiation in this study contrasts with several other studies, which have either reported no increase in lipid-peroxidation products in skin after UV irradiation [21] or an increase only several hours after irradiation [27-29], In these studies the background level of lipid-peroxidation products has been high, even in control, non-irradiated skin. In contrast, in our experiments lipid hydroperoxides were undetectable before irradiation, but appeared at very high concentrations in skin immediately after irradiation. The discrepancies between various studies may be due to differences in technique. The HPLC-chemiluminescence assay used in the present study is specific for lipid hydroperoxides, and has less potential for artifact than the TBARS assay, which is known to suffer from many possibilities for artifact [30] -... 

Chemiluminescence immunoassay These assays rely on the use of chemiluminescent labels, which may be labelled antigens or antibodies. For example, the chemiluminescent label isoluminol is oxidized in the presence of hydrogen peroxide and a catalyst, producing long-lived light emission that is measured, thus allowing determination of the unknown antigen or antibody concentration in a sample. Another useful variant is electrochemiluminescence immunoassay that is commonly used in biomolecular detection, and in particular the measurement of native and recombinant peptides and proteins. 

To demonstrate MT-MEC as a useful platform for protein quantification, a simple surface biotin-avidin assay was constructed[15,16]. In the assay, biotinylated-BSA is incubated on both silvered and glass substrates (Figure 15.5). HRP-streptavidin is then added to the surface, locaiizing the enzyme catalyst in close proximity to the silver for MT-MEC. The peroxide and Acridan (iumophore) are then added to initiate the chemiluminescence reaction. While this assay in essence determines BSA concentration, this model assay could indeed be fashioned to both iocaiize and sense other proteins / DNAs of interest. 

Chemiluminescence offers yet another sensitive detection system, which is easily implemented with simple instrumentation, but suffers to some extent from background interference in complex matrices. A recent example of an enzyme immunoassay with chemiluminescence as the detection system is the assay for 8-oxoguanine in DNA, which uses a secondary antibody conjugated with peroxidase-anti-peroxidase complex and a substrate solution containing hydrogen peroxide, luminol and p-iodophenol. 

Enzymes are nowadays the labels that are most used in immunoassay. Their main disadvantage is their susceptibility to interferences due to changes in assay conditions, e.g., pH, ionic strength, content of organic solvents. The addition of anti-microbial agents such as azides or mercury derivatives also can affect the enzyme activity when using peroxidase labels [96]. The presence of other catalysts in the sample can also have an effect on the enzyme immunoassay result, e.g., Cu(II) ions promote luminol chemiluminescence in the presence of hydrogen peroxide [16]. 

Chemiluminescent labels may be employed in sandwich or competitive antigen assays. In sandwich assays, a solid support holds a primary antibody, and incubation with ligand yields a species that is detectable following a second incubation step with a labeled second antibody. Luminol has been tested as an immunoassay label it may be coupled to proteins through its primary amino group. Luminol reacts with hydrogen peroxide and hydroxide in a microperoxidase-catalyzed reaction, which yields light at 430 nm (Eq. 6.8) ... 

Chemiluminescence methods are known for their high sensitivities. Typical detection limits range from parts per million to parts per billion or lower. Applications include the determination of gases, such as oxides of nitrogen, ozone, and sulfur compounds, determination of inorganic species such as hydrogen peroxide and some metal ions, immunoassay techniques, DNA probe assays, and polymerase chain reacrion methods.- ... 

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