Phthalic acid esters (PAEs) are ubiquitous in the environment, and some of them are recognized as endocrine disruptors that cause concerns on ecosystem functioning and public health. Due to the diversity of PAEs in the environment, there is a vital need to detect the total concentration of PAEs in a timely and low-cost way. To fulfill this requirement, it is highly desired to obtain group-specific PAE binders that are specific to the basic PAE skeleton. In this study, for the first time we have identified the group-specific PAE-binding aptamers via rationally designed target immobilization. The two target immobilization strategies were adopted to display either the phthalic ester group or the alkyl chain, respectively, at the surface of the immobilization matrix. The former enabled the rapid enrichment of aptamers after four rounds of selection. The top 100 sequences are cytosine-rich (44.7%) and differentiate from each other by only 1-4 nucleotides at limited locations. The top two aptamers all display the nanomolar dissociation constants to both the immobilized target and the free PAEs [dibutyl phthalate (DBP), butyl benzyl phthalate (BBP), bis(2-ethylhexyl) phthalate (DEHP)]. We further demonstrate the applications of the aptamers in the development of high-throughput PAE assays and DEHP electrochemical biosensors with exceptional sensitivity [limit of detection (LOD), 10 pM] and selectivity (>10-fold). PAE aptamers targeting one of the most sought for targets thus offer the promise of convenient, low-cost detection of total PAEs. Our study also provides insights on the aptamer selection and sensor development of highly hydrophobic small molecules.

We report here a modified aptamer selection method, magnetic cross-linking precipitation (MCP)-SELEX, for highly efficient library enrichment and aptamer isolation. MCP-SELEX isolates bound aptamers via highly efficient chemical cross-linking between amino groups of target proteins and activated carboxylic acid groups on magnetic beads (>90% coupling efficiency). Importantly, MCP-SELEX avoids surface interferences in conventional target-fixed methods and substantially minimizes nonspecific binding. The enrichment efficiencies of MCP-SELEX for various proteins (PD-L1, ubiquitin, thrombin, and HSA) were all greatly higher than those of the conventional target-bound magnetic bead based-SELEX (MB-SELEX). Antithrombin aptamer with KD of 33 nM was successfully isolated by four rounds of MCP-SELEX. MCP-SELEX also enabled the efficient aptamer isolation by coupling with MB-SELEX or falling-off-SELEX. We identified structure-switching aptamers (SSAs) that specifically bind to HSA with low nanomolar dissociation constant via three rounds of MCP-SELEX and 1 round of falling-off-SELEX. Our HSA SSAs also have ∼3-fold higher specificity against streptavidin relative to thrombin SSAs discovered through falling-off-SELEX only. The enriched library has ∼78-fold higher signal-to-noise ratio (the number of DNAs eluted by 50 nM HSA divided by the number of DNAs self-dissociated in blank buffer) than that obtained by 4 rounds of direct falling-off-SELEX. We finally demonstrated the application of the selected SSA in fluorescent detection of HSA in urine with diagnostic required sensitivity and dynamic range. We expect that MCP-SELEX may be coupled with other selection methods to substantially accelerate aptamer discovery.

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Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the rapid growing of congeners. DNA aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward highly hydrophobic small molecule targets, such as PAEs, is rarely reported. This work describes a bead-based method designed to select group-specific DNA aptamers to PAEs. The amino group functionalized dibutyl phthalate (DBP-NH2) as the anchor target was synthesized and immobilized on the epoxy-activated agarose beads, allowing the display of the phthalic ester group at the surface of the immobilization matrix, and therefore the selection of the group-specific binders. We determined the dissociation constants of the aptamer candidates by quantitative polymerization chain reaction coupled with magnetic separation. The relative affinities and selectivity of the aptamers to other PAEs were determined by the competitive assays, where the aptamer candidates were pre-bounded to the DBP-NH2 attached magnetic beads and released to the supernatant upon incubation with the tested PAEs or other potential interfering substances. The competitive assay was applied because it provided a facile affinity comparison among PAEs that had no functional groups for surface immobilization. Finally, we demonstrated the fabrication of an electrochemical aptasensor and used it for ultrasensitive and selective detection of bis(2-ethylhexyl) phthalate. This protocol provides insights for the aptamer discovery of other hydrophobic small molecules.

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