Development of an aptasensor for dibutyl phthalate detection and the elucidation of assay inhibition factors

Abstract

We developed a fluorescence aptasensor (hereafter ‘SG-aptasensor’) using SYBR Green I, a newly truncated 20-mer aptamer, and probe DNA to detect dibutyl phthalate (DBP). The detection range of DBP was 0.1–100 ng L⁻¹ with 0.08 ng L⁻¹ as the limit of detection. To adapt the assay to environmental samples in the near future, possible inhibition factors (experimental and environmental) have been tested and reported. The experimental inhibitors included the incubation time, temperature, pH, and ionic strength. Consequently, temperature (2–25 °C) and pH (7.0–9.0) ranges did not significantly inhibit the assay. The incubation time required for sufficient reaction was at least 4 h, and a relative humidity <20% may have induced fluorescence quenching. Tris–HCl-based incubation buffer with excess ionic strength (more than 0.2 M NaCl) demonstrated an abnormal increase in fluorescence. Environmental inhibitors including cations (Mg²⁺, Ca²⁺, and Cu²⁺) and humic acids were tested. The fluorescence signal was significantly reduced (∼99%) by 100 mM Cu²⁺ compared to that by 0 mM Cu²⁺. In contrast, the reduction in fluorescence signal was marginal (<15%) when Mg²⁺ or Ca²⁺ ions were present. Inhibition of the assay was observed (∼28%) in the presence of 100 mg L⁻¹ humic acids.