Development of an Aptamer-Based qPCR Method for the Selective and Rapid Picomolar-Level Detection of Perfluorooctanesulfonic Acid in Water

Abstract

Per- and polyfluoroalkyl substances (PFAS) are widely recognized as emerging contaminants because they are ubiquitous in various environmental media. Their potential for chronic toxicity after prolonged human exposure is a growing concern. Consequently, there is an urgent need to develop an appropriate technology to efficiently treat and rapidly and consistently monitor PFAS levels. This study reports the development of the first aptamers that can bind to perfluorooctanesulfonic acid (PFOS), with a dissociation constant (K_D) of 6.76 μM, and exhibit a high specificity for PFOS even in the presence of other PFAS. The binding site and mechanism of the prepared aptamers are explored using truncation and molecular dynamics simulations, which show that the lengths of fluorocarbons and functional groups are important recognition epitopes. To demonstrate the application potential of the prepared aptamers, an aptamer-based quantitative polymerase chain reaction method is also developed, which exhibits picomolar-level detection capabilities and a limit of detection of 5.8 pM (2.9 ng/L), indicating its high sensitivity. Our findings demonstrate the potential of the developed method in the rapid in situ monitoring of PFOS at contamination sites, which will facilitate its early detection before rigorous analysis.

Keywords: aptamer discovery; aptamer-based sensing method; molecular dynamics simulation; perfluorooctanesulfonic acid; qPCR detection.

1

Comparison of the fluorescence responses of different aptamer candidates for (A) 25 μM PFOS and a blank solution and (B) 25 μM PFOS with and without mixed countermolecules. (C) Changes in the fluorescence responses of the PFOS_JYP_2 aptamer for a blank solution and PFOS, PFNA, PFOA, PFHxS, octanesulfonic acid, PFBS, PFBA, and PFDA with concentrations of 1.563–50 μM with 1:2 serial dilution. For all experiments, the conditions were as follows: aptamer concentration = 400 nM, ThT concentration = 10 μM, and pH = 7.5. Error bars represent the standard deviation of experimental triplicates.

2

(A) Secondary structures PFOS_JYP_1 and PFOS_JYP_2 predicted using the MFold web-based program. The inset table shows the phylogenetic tree and sequence homology analyses of PFOS_JYP_1 and PFOS_JYP_2 (consensus sequences are marked in red) performed using MultAlin. (B) Changes in the fluorescence responses of PFOS_JYP_2 and the upper loop at a pH of 7.5. Herein, 0–50 μM PFOS with 1:2 serial dilution was incubated with each aptamer (400 nM) and 10 μM ThT. Error bars represent the standard deviation of experimental triplicates. (C) Predicted 3D structures of the upper small loop shown in (B) and 16T aptamers. Distances are marked in red (Å), and guanine at the ninth position is marked using a red circle.

3

MDS results showing the binding sites of PFOS_JYP_2 for PFOS, PFNA, PFHxS, PFOA, PFBS, and octanesulfonics and the involved chemical interactions. Green, cyan, red, and yellow dashed lines represent conventional hydrogen, halogen, nonclassical hydrogen (C–H), and π–sulfur bonds, respectively.

4

(A) Proposed design and mechanism of the qPCR-based aptasensor and (B) changes in the Ct responses in the PFOS concentration range of 0–400 pM with 1:2 serial dilution for 100 pmol of PFOS_JYP_6. The Hill’s (black line) and linear (red-dashed line) fits were used for fitting. The solution pH was maintained at 7.5. Error bars represent the standard deviation of experimental triplicates.