Redox-based assays

A major drawback of redox-based assays is that any oxidizable compound in the sample solution will react, too. Ascorbic acid, sulphur dioxide (in wines), aromatic amines and carbohydrates (production of endiol reductones in alkaline solution) are the most frequently encountered reductants [47,106]. 

Metal complexing assays (i.e. Jerumanis method) are generally more specific than redox-based assays, because the color of the complexes depends on a specific pattern of substitution on the phenolic rings [108]. Procyanidins form chelates with metal ions via their ortho-diphenolic groups on the B-rings [112],... 

ET-based assays generally set a fixed time for the concerned redox reaction and measure thermodynamic conversion (oxidation) during that period. Although the reducing capacity of a sample is not directly related to its radical scavenging capability, it is a very important parameter of antioxidants (Apak et al. 2013). 

However, the rate of scavenging of ARTS radicals by weak AOXs that have higher redox potentials or form stable intermediates was found to be very slow. This suggests that an ARTS -based assay may be selective for active AOXs in the presence of weak or inactive substances. 

Because of the time and expense involved, biological assays are used primarily for research purposes. The first chemical method for assaying L-ascorbic acid was the titration with 2,6-dichlorophenolindophenol solution (76). This method is not appHcable in the presence of a variety of interfering substances, eg, reduced metal ions, sulfites, tannins, or colored dyes. This 2,6-dichlorophenolindophenol method and other chemical and physiochemical methods are based on the reducing character of L-ascorbic acid (77). Colorimetric reactions with metal ions as weU as other redox systems, eg, potassium hexacyanoferrate(III), methylene blue, chloramine, etc, have been used for the assay, but they are unspecific because of interferences from a large number of reducing substances contained in foods and natural products (78). These methods have been used extensively in fish research (79). A specific photometric method for the assay of vitamin C in biological samples is based on the oxidation of ascorbic acid to dehydroascorbic acid with 2,4-dinitrophenylhydrazine (80). In the microfluorometric method, ascorbic acid is oxidized to dehydroascorbic acid in the presence of charcoal. The oxidized form is reacted with o-phenylenediamine to produce a fluorescent compound that is detected with an excitation maximum of ca 350 nm and an emission maximum of ca 430 nm (81). 

The possibility of isolating the components of the two above-reported coupled reactions offered a new analytical way to determine NADH, FMN, aldehydes, or oxygen. Methods based on NAD(P)H determination have been available for some time and NAD(H)-, NADP(H)-, NAD(P)-dependent enzymes and their substrates were measured by using bioluminescent assays. The high redox potential of the couple NAD+/NADH tended to limit the applications of dehydrogenases in coupled assay, as equilibrium does not favor NADH formation. Moreover, the various reagents are not all perfectly stable in all conditions. Examples of the enzymes and substrates determined by using the bacterial luciferase and the NAD(P)H FMN oxidoreductase, also coupled to other enzymes, are listed in Table 5. 

MesoScale Discovery (MSD) succeeded in introducing product with a similar technology approach based upon ruthenium redox-mediated electrochemical detection (Figure 2.14). MSD is a joint venture of its parent company, MesoScale, and IGEN, a company that pioneered much of fhe work on electrochemical detechon based on the ruthenium redox system. MSD s Multi-Spot plates contain antibodies immobilized on multiple working electrode pads within each well, allowing each spot within the well to serve as an individual assay. Multiplexed cytokine immxmoassays can be performed in 96-well (4,7, or 10 spots per well) patterns with detection limits of 1 to 10 pg/mL and a linear dynamic range up to 3,000 pg/mL. Both 24-and 384-well electrode systems are available. 

In the Progress Rept No 20-311 (Ref 21) are given the following procedures a)Assay of AN by a Redox Titration with Alkali Hy-pobromite Solution [This method is based on the method described by G. M. Arcand ... 

Although multistep affinity assays with redox-labeled targets have been described (Wang et al. [117]), most of the assays use enzyme-labeled species in conventional indirect formats (competitive, non-competitive). Direct EILAs based on multistep electrochemical affinity assays have also been developed with excellent results. In all these cases the MIP is used to extract the analyte from the sample and, after elution, the analyte is carried on to the electrochemical flow-through cell for being detected. 

Immunoassays, electrochemical — A quantitative or qualitative assay based on the highly selective antibody-antigen binding and electrochemical detection. Poten-tiometric, capacitive, and voltammetric methods are used to detect the immunoreaction, either directly without a label or indirectly with a label compound. The majority of electrochemical immunoassays are based on -> voltammetry (-> amperometry) and detection of redox-active or enzyme labels of one of the immunochemical reaction partners. The assay formats are competitive and noncompetitive (see also -> ELISA). 

Next, the one-electron reduction of superoxide to H2O2 is indeed energetically easy. The redox potential for 02I0 2 ) implies however that superoxide is also a reasonably efficient one-electron reductant and we will see that kinetic constraints imply that free 0 2 will usually behave as a reductant. This property has been widely used for the popular assay of superoxide based on the reduction of ferric cytochrome C [36], which is associated with a bimolecular rate constant of 2.6xl05M-1 s-1. 

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