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.