A simple, post-additional antioxidant capacity assay using adenosine triphosphate-stabilized 2,2′-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radical cation in a G-quadruplex DNAzyme catalyzed ABTS–H2O2 system

The scavenging of 2,2′-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radical cation (ABTS+) by antioxidants has been widely used in antioxidant capacity assay. Because of ABTS+ disproportionation, however, this radical cannot be prepared on a large scale and stored long-term, making it unsuitable for high-throughput detection and screening of antioxidants. We developed a modified “post-additional” antioxidant capacity assay. This method possessed two remarkable features: First, instead of natural peroxidases, an artificial enzyme, G-quadruplex DNAzyme, was used for the preparation of ABTS+, thus greatly reducing the cost of the assay, and eliminating the strict demand for the storage of enzymes. Second, an ABTS+ stabilizer, adenosine triphosphate (ATP), was used. In the presence of ATP, the disproportionation of ABTS+ was effectively inhibited, and the lifetime of this radical cation was prolonged about 6-fold (12 days versus 2 days), making the large-scale preparation of ABTS+ possible. Utilizing this method, the antioxidant capacities of individual antioxidants and real samples can be quantified and compared easily. In addition, this method can be developed as a high-throughput screening method for antioxidants. The screening results could even be judged by the naked eye, eliminating the need for expensive instruments.

► A simple “post-additional” method for antioxidant capacity determination is developed.
► A cheap and stable artificial enzyme, G-quadruplex DNAzyme, is used instead of natural peroxidases.
► The stabilization of the pre-formed ABTS+ by ATP makes the large-scale preparation of ABTS+ possible.
► Using this method, the relative antioxidant capacity of antioxidants can be detected and compared easily.
► This method can be used for high-throughput visual screen of antioxidants.