Sensitive Silver-Enhanced Microplate Apta-Enzyme Assay of Sb³⁺ Ions in Drinking and Natural Waters

The toxic effects of antimony pose risks to human health. Therefore, simple analytical techniques for its widescale monitoring in water sources are in demand. In this study, a sensitive microplate apta-enzyme assay for Sb³⁺ detection was developed. The biotinylated aptamer A₁₀ was hybridized with its complementary biotinylated oligonucleotide T₁₀ and then immobilized on the surface of polysterene microplate wells. Streptavidin labeled with horseradish peroxidase (HRP) bound to the biotin of a complementary complex and transformed the 3,3',5,5'-tetramethylbenzidine substrate, generating an optical signal. Sb³⁺ presenting in the sample bounded to an A₁₀ aptamer, thus releasing T₁₀, preventing streptavidin-HRP binding and, as a result, reducing the optical signal. This effect allowed for the detection of Sb³⁺ with a working range from 0.09 to 2.3 µg/mL and detection limit of 42 ng/mL. It was established that the presence of Ag⁺ at the stage of A₁₀/T₁₀ complex formation promoted dehybridization of the aptamer A₁₀ and the formation of the A₁₀/Sb³⁺ complex. The working range of the Ag⁺-enhanced microplate apta-enzyme assay for Sb³⁺ was determined to be 8-135 ng/mL, with a detection limit of 1.9 ng/mL. The proposed enhanced approach demonstrated excellent selectivity against other cations/anions, and its practical applicability was confirmed through an analysis of drinking and spring water samples with recoveries of Sb³⁺ in the range of 109.0-126.2% and 99.6-106.1%, respectively.