Preliminary Establishment Of Single-Molecule Monitoring Methods For Per-and Polyfluorocarboxylic Acids

The treatment of New Pollutants is a brand-new and arduous mission faced by Ecological Civilization Construction during the 14th Five-Year Plan period.The monitoring of New Pollutants is the important foundation for scientific governance work.As a typical type of New Pollutants,per-and polyfluoroalkyl substances(PFASs)have attracted global attention due to their significant bioaccumulation and physiological toxicity.Perfluorocarboxylic acids(PFCAs)are a type of PFASs that is under key control.Currently,it has been reported that there are approximately 9000 to 12000 homologues of PFCAs and chemical compounds with similar structures,and different structures may correspond to vastly different environmental behaviors as well as physical and chemical properties.Therefore,accurate monitoring of the occurrence of conventional and emerging PFCAs in different environmental media is crucial for understanding their internal and external exposure pathways.At present,liquid chromatography-tandem mass spectrometry is still the most common method for quantitative analysis of conventional PFCAs,and high-resolution mass spectrometry is used for non-targeted identification of various per-and polyfluorocarboxylic acids with unknown structures.However,the liquid chromatography-mass spectrometry technique is limited by the quantity of authentic standards and can hardly meet the demand of high-throughput screening;while the high-resolution mass spectrometry is limited by spectral libraries and other limitations,resulting in differences in data from different analysis platforms,thus it can hardly support quantitative analysis effectively.Therefore,existing monitoring techniques are unable to meet the monitoring demands of a wide range of per-and polyfluorocarboxylic acids,and it’s urgent to develop a new technique for screening per-and polyfluorocarboxylic acids that can carry out quantitative analysis at the same time.In addition,quantifying target pollutants of trace environmental concentrations based on chromatography and mass spectrometry methods is easily affected by interfering substances of high concentrations.In order to overcome the problem of excessive reliance on authentic standards and difficulty in quantifying trace but critical target pollutants under interferences of high concentrations in traditional methods for monitoring per-and polyfluorocarboxylic acids,this study developed a novel method for monitoring per-and polyfluorocarboxylic acids based on nanopore sensing.Single-molecule monitoring based on nanopore electrochemistry can not only identify trace but critical analytes in complex systems by measuring target molecules individually and calculating their exact quantities,but also accurately quantify them without calibration.The main results are as follows:(1)Taking straight-chain PFCAs with different chain lengths(C5,C6,C7)as examples,the feasibility of single-molecule monitoring of perfluorocarboxylic acids was verified and a basic method was preliminarily established,which applied Aerolysin nanopore to detect PFCAs conjugated to polycationic peptide probes.By studying the influence of electrolyte concentration on key parameters of nanopore-based single-molecule monitoring as well as qualitative and quantitative detection performances,4 M KCl was determined to be the optimal electrolyte condition;(2)The influences of electrolyte concentration,probe type,and applied voltage on the construction of the linear relationship between current blockade and molecular volume of perfluorocarboxylic acids were studied,and the optimal experimental conditions(4 M KCl,R6 probe,and-50 mV)were determined.Single-molecule monitoring was performed on all 9 straight-chain PFCAs(C0 to C9),and the linear relationship between current blockade and molecular volume of per-and polyfluorocarboxylic acids was constructed based on nanopore electrochemistry;(3)Taking 5 hydrogenated or chlorinated polyfluorocarboxylic acids(3H,3C1,5H,FTA,7H)as examples,it was verified that the linear relationship between current blockade and molecular volume can predict any unknown structure of per-and polyfluorocarboxylic acids within the linear range with no need of authentic standards,with prediction accuracies of nearly 100%.Based on the inherent advantage of multiple characteristic parameters of single-molecule signals,the single-dimensional recognition based on current blockade is extended to multidimensional recognition based on current blockade,dwell time,and standard deviation of current blockade.Combining machine learning,single-molecule algorithm(SMA)is designed.By comparing the quantification accuracies of traditional multi-peaks Gaussian fitting and SMA in quantifying trace but critical pollutants under the interference of high concentration,the quantitative advantage of single-molecule monitoring of per-and polyfluorocarboxylic acids was verified.In addition,the recognition accuracy of 95.1%was achieved for all 14 per-and polyfluorocarboxylic acids within the linear range with the help of SMA.The quantitative method for single-molecule monitoring of per-and polyfluorocarboxylic acids was demonstrated through the linear relationship between the interval time of single-molecule signals and the concentration of analytes,and two strategies were proposed to reduce the limit of detection:increasing the system temperature and increasing the applied voltage.In summary,single-molecule monitoring of per-and polyfluorocarboxylic acids based on nanopore electrochemistry has unique qualitative and quantitative detection advantages,which can meet the demands of rapid and quantitative on-site screening of per-and polyfluorocarboxylic acids,and be effectively complementary to traditional monitoring techniques.