Construction of a multiplex biosensor for the on-site rapid detection of phthalates in biological fluids based on single-stranded DNA aptamers.

Current analytical methodologies for phthalic acid esters (PAEs) face significant challenges in achieving rapid on-site detection and the discriminative identification of individual congeners. This study utilizes Capture-SELEX technology to identify aptamers specifically recognizing dibutyl phthalate (DBP), di-n-octyl phthalate (DNOP), and diethyl phthalate (DEP). Fluorescence-based affinity assays revealed that these aptamers possess dissociation constants (Kd) of 1.577, 0.889, and 0.173 μM for their respective targets. Molecular docking simulations, coupled with base mutation analyses, were conducted to predict potential binding interfaces. By integrating these aptamers with the previously characterized di(2-ethylhexyl) phthalate (DEHP)-aptamer, a gold nanoparticle (AuNPs)-mediated visual detection platform was developed to validate the efficacy and potential of these aptamers for subsequent applications. The calculated limits of detection (LOD) for DBP, DEHP, DNOP, and DEP using this system are 17.64, 2.32, 25.68 and 22.01 ng/mL, respectively. Furthermore, a lateral flow strip was designed to enable the simultaneous and on-site multiplex detection of the four PAEs. This strip achieved LODs of 12.54, 14.95, 14.93 and 30.28 ng/mL for DBP, DEHP, DNOP, and DEP, respectively. A comparative evaluation using LC-MS on identical samples revealed quantitative concordance within a ratio range of 94.18 %-110.43 %, highlighting the robust analytical performance, detection efficiency, and translational potential of the strip for widespread application in various PAE testing scenarios.