Resistivity detection of perfluoroalkyl substances with fluorous polyaniline in an electrical lateral flow sensor

Significance

Lateral flow assays (LFAS) have garnered a broad public acceptance for sensing in healthcare and offer expanded applications for environmental sensing. With the increasing concern for the widespread detection of per- and poly-fluoroalkyl substances (PFAS), selective sensing is imperative for monitoring these harmful chemicals in the environment and drinking water. We report a low-cost and sensitive detection method for quantifying PFAS with an e-LFA (electrically read lateral flow assay). PFAS dopes the polyaniline (PANI) to increase conductivity and enable e-LFA detection. Our method harnesses the fluorous effect to selectively target perfluoroalkyl acids over their nonfluorous equivalents. Our F-PANI (fluorous PANI) fabricated e-LFA exhibits a 400-ppt detection limit for PFOA (perfluorooctanoic acid) and provides quantitative measurements from simple resistivity measurements.

Abstract

Perfluoroalkyl substances (PFAS), known as “forever chemicals,” are a growing concern in the sphere of human and environmental health. In response, rapid, reproducible, and inexpensive methods for PFAS detection in the environment and home water supplies are needed. We have developed a simple and inexpensive perfluoroalkyl acid detection method based on an electrically read lateral flow assay (e-LFA). Our method employs a fluorous surfactant formulation with undoped polyaniline (F-PANI) fabricated to create test lines for the lateral flow assay. In perfluoroalkyl acid sensing studies, an increase in conductivity of the F-PANI film is caused by acidification and doping of PANI. A conductivity enhancement by 10⁴-fold can be produced by this method, and we demonstrate a limit of detection for perfluorooctanoic acid (PFOA) of 400 ppt and perfluorobutanoic acid of 200 ppt. This method for PFOA detection can be expanded for wide-scale environmental and at-home water testing.

PFAS: Per- and poly-fluoroalkyl substances (PFAS) contain fully fluorinated alkyl groups and have been widely used to provide waterproof, anti-stain, and heat-resistance properties (1, 2). However, their extraordinary stability has allowed for accumulation in water supplies, and this is now recognized as a serious threat to public health (3, 4). Studies show that PFAS may result in adverse effects including increased cholesterol levels, thyroid disease, liver damage, kidney cancer, testicular cancer, developmental effects affecting the unborn child, and other environmental damage (1, 5, 6). In response to this issue, the US Environmental Protection Agency (EPA) has introduced regulatory guidance to limit the amount of six different PFAS in drinking water with levels of 4 ppt (4 ng L^–1) for perfluorooctanoic acid (PFOA) and 1 ppt for perfluorobutanoic acid (PFBA) in March 2023 (7). Currently, the EPA employs liquid chromatography and mass spectrometry for PFAS detection at the ng L⁻¹ level (8). Yet, these methods are time-consuming, expensive, require well-trained personnel, and must be performed in laboratory environments (8, 9). To facilitate broader testing and source attribution, fast, portable, user-friendly, and low-cost PFAS sensing methods are needed that robustly meet the EPA-required ppt detection limits.

We report herein a PFAS sensing platform based on the conducting polymer, polyaniline (PANI), and electrical lateral flow assay (e-LFA, Fig. 1). PANI is an attractive material to create sensors, as a result of its facile synthesis, high stability, and large conductivity changes caused by protonic doping as described in Fig. 1A (10, 11). To target PFAS-responsive polymer coatings, we mixed the fluorous surfactant (Krytox-PEG-600-diamide, KPD) (12) with emeraldine free-base (PANI-EB) state by ultrasonication in water to create a dispersion as shown in Fig. 1B. Therefore, we yield a material that selectively absorbs PFAS as a result of fluorous interactions (13, 14). In the case that the functional groups of PFAS contain acidic functionality, absorption will result in acidification of the film and a transition of the PANI from its insulating emeraldine free-base (PANI-EB) state to highly conductive emeraldine salt (PANI-ES) polymer (Fig. 1C). To create responsive sensory devices, we print a KPD-(PANI-EB) test line on nitrocellulose (NC) membranes. Aqueous perfluoroalkyl acid solutions move by capillary force along the NC membrane, and a calibrated amount of water transverses the conducting polymer test line to produce an economical, fast, quantitative, and easy-to-use flow assay (15–17).

Fig. 1.

A PFAS sensing platform based on PANI polymer and e-LFA. (A) Doped and undoped PANI structures. (B) Preparation of fluorous PANI-EB dispersion by mixing PANI-EB nanofiber powder with the fluorous surfactant (KPD). (C) Photograph of the e-LFA membrane and schematic of the conductivity change after exposure to PFAS aqueous solution.

Results and Discussion

Preparation of Polyaniline Nanofibers, Ink, and Test Lines.

PANI is polymerized in nanofiber form to ensure a high surface area to enhance analyte interactions. Nanofibers with diameters around 80 nm were produced by an interfacial oxidative polymerization method using camphorsulfonic acid and ammonium persulfate (SI Appendix, Fig. S1) (10, 18). The synthesized PANI-ES powder is then converted to undoped PANI-EB (emeraldine base) by treatment (washing then filtration) with ammonium hydroxide solution. PANI-EB formation is confirmed by Fourier-transform infrared spectroscopy (FTIR) with a quinoid ring stretching at 1,576 cm⁻¹, benzenoid ring stretching at 1,491 cm⁻¹, and C–N stretching modes at 1,378 cm⁻¹ and 1,295 cm⁻¹ (SI Appendix, Fig. S1) (19, 20).

The Fluorous-PANI-EB (F-PANI) ink is prepared by mixing PANI-EB dispersion with KPD solution and sonicating for 30 min (Fig. 1B). The ink is likely stabilized by the noncovalent interactions between KPD (amide and ether groups) and the nitrogen atoms of PANI-EB as shown schematically in Fig. 1B. Deposition of F-PANI ink on NC membrane or filter paper substrates resulted in test line bands. Detailed procedures for the preparation of the materials and test lines are described in the Materials and Method section.

Scanning electron microscope (SEM) images reveal microcracks in the surface of coatings formed on the NC membrane (Fig. 2 A and B). The dehydration of PANI likely provides the stresses that result in the microcracks that are not apparent with visual inspection (10). Microcracks still remained in the wet F-PANI coating (SI Appendix, Fig. S9). The rehydration and swelling of the materials likely occur during the sensing experiments, and although the microcracks can produce unwanted resistance in the test lines, they may also provide for expanded interactions with the aqueous solutions for improved partitioning of the analytes into the films. The crack widths of the F-PANI coating are ca. 3 μm, which is smaller than those observed in the coating (ca. 15 μm) produced from pure PANI-EB inks. We attribute that the fluorous surfactant prevents aggregation between PANIs and hence favors smaller features. The F-PANI is expected to be less hydrophilic than PANI-EB, and this was evaluated by measuring contact angles (θ) of water droplets placed on the films (Fig. 2C). We confirmed that the F-PANI coating presents a hydrophobic surface regardless of the substrate, whereas the PANI-EB coating surface is hydrophilic.

Fig. 2.

Characterization of PANI coatings SEM images of (A) PANI-EB and (B) F-PANI coatings formed on a NC membrane. (C) Plots of contact angle (θ) of water on PANI-EB and F-PANI coatings formed on the NC membrane and glass. Insets are optical microscopic images of the droplet contact angles. Each data point was averaged from 10 measurements, and the error bars correspond to the SD.

Factors that Affect the Conductivity of Test Lines.

Resistance measurements were collected with a four-point probe. Multiple measurements on each test line were performed to investigate the uniformity of the materials. As shown in Fig. 3A, the conductivity is influenced by multiple different factors. The resistance of the F-PANI test line decreases after exposure to aqueous solutions of PFOA (Fig. 3B). Hydration of the test line is important, and if a test strip is removed from the solution and air-dried for 15 min, the water evaporates and the test lines display a high resistance >220 MΩ, which is the limit of our detection. This feature is due to the cationic (polaron) and dicationic (bipolaron) carriers that are pinned by attractive electrostatic interactions with the counterions in the absence of water (21). Water reduces these interactions by a solvation of the ions/carriers by a combination of screening and separation of the charges. To avoid dehydration, resistivity measurements were made within 5 min after the sample was removed from the vial to ensure full hydration.

Fig. 3.

Resistance and conductivity measurement of the F-PANI test line in the e-LFA using the four-point probe system. (A) Schematic of the four-point probe measurement and experimental factors investigated. (B) Plot of resistance value as a function of time after the sample was removed from the vial. (C) Plot of resistance value as a function of exposure time in the vial with 10⁻⁶ M PFOA. Resistance values were collected from over 30 different locations with 3 samples for each concentration. (D and E) Plot of resistance values for each width with 10⁻⁶ M PFOA (D) and 10⁻² M PFOA (E). The distance from the end of the test line to the end of the NC membrane was 1.6 cm for all devices investigated.

The absorption of perfluoroalkyl acids into the polymer coating is critical to obtaining an optimal response, and this is facilitated by the fact that the solution passes very slowly through the hydrophobic F-PANI test line (Fig. 2C). Solutions require 20 min to completely pass through the F-PANI test line, whereas only 90 s is required with an equivalent PANI-EB test (SI Appendix, Fig. S2). Fig. 3C shows that the test line reaches a constant resistance value of approximately 67 MΩ after 30 min of being dipped into a 10⁻⁶ M PFOA solution. Hence, the main time limitation is the transport of the water along the NC membrane, and the absorption of the PFBA and PFOA with concurrent protonic doping of F-PANI is a relatively rapid process. Therefore, resistivity measurements were made after 30 min of the test strip being placed in the solution.

The width of the F-PANI test line was investigated. Lines with widths of 0.3 cm, 0.7 cm, and 1.4 cm were created using 17 µL, 40 µL, and 80 µL of F-PANI ink, respectively. They had similar thicknesses (27.9 ± 5.9 µm). It was found that test lines 0.7 cm or less provided consistent results over a range of analyte concentrations (Fig. 3 D and E). Wider test lines (1.4 cm) displayed higher resistances with 10⁻² M PFOA and large SD (86.7 ± 79.7 kΩ) as compared to the 0.7-cm lines (30.7 ± 17.5 kΩ). It is likely that in these cases, protonic doping is not uniform throughout the test line. The data for other concentrations are given in the SI Appendix, Table S1 and are consistent with the previous results. As a result, test line widths of 0.7 cm were used to determine the sensor performance.

Ultratrace PFAS Detection.

The performance of the F-PANI lateral flow devices was evaluated for PFOA detection. The resistance measurements are limited to values less than 220 MΩ, which is the resistance of the assay with PFAS-free water. The histogram of resistance values of F-PANI test lines on the NC membrane for each concentration of PFOA is shown in Fig. 4A. Because of nonuniformity in the films, the resistance for each concentration taken with a colinear 4-point probe has a distribution rather than a single clear value. The distribution could potentially be the result of microcracks of the coating that can complicate the conductive pathways and also from nonuniform protonic doping of the PANI backbone. For the statistical analysis of the data, we fit our data to single Gaussian functions to obtain the peak value of resistance (R_M) for each concentration and its SD (σ_M). We emphasize that no data are omitted. This method allows us to avoid bias in the data by excluding outliers (22). The detection limit of an analyte is the concentration at which the value obtained by adding the SD (σ_M) to the peak resistance value (R_M) acquired at a certain concentration begins to fall below 220 MΩ which is the resistance of the assay with PFAS-free water as the concentration increases. For PFOA, the detection limit is 400 ppt (10⁻⁹ M), and the change in conductivity increases by 10⁴-fold for a 10⁻² M PFOA concentration. Table 1 summarizes the resistance and conductivity values of the F-PANI test lines on NC membranes as a function of PFOA concentration. Similarly, detailed data for OA are given in the SI Appendix, Fig. S3 and Table S2.

Fig. 4.

Resistance and conductivity of wet F-PANI coatings. (A) The histogram of resistance values for each concentration of PFOA. The bin size of the histogram was decreased at lower concentrations. The values were collected from over 30 different locations with 3 samples for each concentration. The measurement of the wet coating was carried out within 5 min after the sample was removed from the vial. (B) Plots of conductivity of F-PANI coatings on the NC membrane as a function of PFOA (black spheres) or OA (green square) concentrations. The point of 10⁻² M OA is not indicated because it is not a uniform solution in ambient conditions. (C) Plots of conductivity of F-PANI coatings on NC membrane (black spheres) and filter paper (green triangles) substrates as a function of PFOA concentrations.

Table 1.

Summary of resistance and conductivity values of the wet F-PANI test line on the NC membrane as a function of PFOA concentration (data from Fig. 4 A and B)

PFOA [M]	RA ± σA [MΩ, kΩ for §]	RM ± σM [MΩ, kΩ for §]	CM [S/m]	Conductivity enhancement (times)
0	>220	>220	<3.70 • 10−5	-
10−10	205.9 ± 27.4	218.5 ± 8.2	3.73 • 10−5	>1.01
10−9	191.4 ± 24.3	202.5 ± 10.0	4.03 • 10−5	>1.09
10−8	149.8 ± 25.0	154.5 ± 23.0	5.27 • 10−5	>1.42
10−7	113.9 ± 29.8	110.3 ± 21.7	7.42 • 10−5	>2.01
10−6	75.7 ± 27.9	69.7 ± 18.6	1.17 • 10−4	>3.16
10−5	43.8 ± 21.3	36.1 ± 17.9	2.26 • 10−4	>6.11
10−4	7.6 ± 5.9	5.6 ± 3.4	1.46 • 10−3	>3.95 • 101
10−3	1.2 ± 2.1	318.9 ± 89.2 §	2.56 • 10−2	>6.92 • 102
10−2	22.0 ± 7.3 §	23.7 ± 6.7 §	3.44 • 10−1	>9.30 • 103

§R_A and σ_A indicate average and sigma (SD) values, respectively. §R_M and σ_M indicate mean (peak) and sigma values estimated from Gaussian fitting curves. C_M is the conductivity calculated from the R_M value. 220 MΩ is the maximum resistance value that can be measured.

Fig. 4B shows the plots of conductivity of F-PANI test lines on the NC membrane as a function of PFOA and its nonfluorous form (OA) concentrations obtained by Gaussian fitting of the histogram of resistance values. The aqueous PFOA analyte shows higher conductivity compared to OA at the same concentration.

We also tested the effect of different substrates. Fig. 4C shows the plots of conductivity of F-PANI test lines on NC membrane and filter paper substrates as a function of PFOA concentrations. The conductivity responses are independent of the substrate and correlate with the PFOA concentration. The data for the F-PANI test line on the filter paper are described in the SI Appendix, Fig. S4 and Table S3. The fluorous nature of the F-PANI is critical, and a flow assay using PANI-EB is 10⁵ times less sensitive to PFOA with a detection limit of 10⁻⁴ M (SI Appendix, Fig. S5 and Table S4). We also found that the performance of the F-PANI test lines was the same when the aqueous media were changed from D.I. water to our local (Cambridge, MA) tap water. (SI Appendix, Fig. S6 and Table S5). The F-PANI lateral flow assay is also capable of detecting PFBA at 10⁻⁹ M (200 ppt) similar to the PFOA limits of detection (SI Appendix, Fig. S7 and Table S6). Here again, we have high selectivity for the fluorous acid over the nonfluorous equivalent, butyric acid, which gives responses that are 10⁴ times lower (SI Appendix, Fig. S8 and Table S7).

Conclusion

We have developed simple e-LFAs for the detection of PFOA with limits of detection down to 400 ppt. Although additional optimization and larger sample sizes than those used in the current e-LFA are necessary to align with the current US EPA limits, the remarkably low detection thresholds of this sensor scheme render it promising for on-site PFAS detection. Our transduction method is the protonic acid doping of polyaniline in its insulating emeraldine base form (PANI-EB) to produce an electrical conducting PANI-ES. A formulation of PANI-EB and a fluorous polymeric surfactant creates a conducting polymer with a fluorous character (F-PANI). Test lines of F-PANI are printed on NC membranes or filter paper, and the wicking of aqueous solutions results in the flow of water through the polymer test line. When PFOA is present, it is absorbed into the F-PANI and acidifies the film resulting in protonic doping of the PANI to create charge carriers. This simple, inexpensive, rapid, and quantitative PFAS detection method is ideally suited for monitoring PFAS in areas, such as military bases, airports, and industrial locations where PFAS exposure is a concern (23).

Materials and methods

Materials and Characterization.

Aniline (ACS reagent, ≥99.5%), ammonium persulfate (ACS reagent, ≥98%), and camphorsulfonic acid (≥98%) were purchased from Sigma-Aldrich and used as received. The fluorous surfactant, KPD, was synthesized by the previously reported procedure and purchased from Akita Innovations LLC (12). All solvents used were of High-Performance Liquid Chromatography (HPLC) grade. All aqueous solutions were prepared, and samples were rinsed using Milli-Q water. The NC membrane strip with a polyester backing card was purchased from GE Healthcare Life Sciences. The filter paper (494, Quantitative) for the substrate was purchased from VWR International.

Scanning Electron Microscope (SEM) images of the films were obtained by a Merlin and Crossbeam 540 Zeiss scanning electron microscope. The Transmission Electron Microscope (TEM) images of nanofibers were obtained with a 120-kV FEI Tecnai multipurpose transmission electron microscope. A Thermo Scientific Nicolet 6700 FT-IR instrument with a Ge crystal was used to obtain attenuated total reflectance FTIR spectra. Resistivity measurements were conducted with a Keithley 2400 and a Signatone four-point probe. The thickness of the coatings on the NC membrane was obtained with a Dektak 6M stylus profilometer.

Synthesis of PANI Polymer.

The PANI nanofiber was synthesized via the interfacial polymerization of aniline (SI Appendix, Fig. S1). Distilled aniline was used for experiments. The polymerization was performed in a 20-mL glass vial. Aniline (3.2 mmol) was dissolved in 10 mL of dichloromethane to yield the organic phase (10, 18). Then, 0.8 mmol of ammonium persulfate was dissolved in 10 mL of 1 M camphorsulfonic acid solution to yield the aqueous phase. The interfacial polymerization of aniline was targeted by combining the aqueous and organic phases into a 20-mL vial. After 14 h (o/n), the as-prepared PANI-ES was purified by filtration. PANI-EB (Emeraldine base) form was obtained by reduction with a 0.1 M aq. NH₄OH solution. SI Appendix, Fig. S1 shows Fourier-transform infrared spectra of PANI-EB nanofibers. The FTIR spectrum displays quinoid ring (Q) stretching at 1,576 cm⁻¹, benzenoid ring (B) stretching at 1,491 cm⁻¹, C–N stretching vibration near quinone diimine unit at 1,378 cm⁻¹, C–N stretching in cis-Q-B-Q, Q-B-B and B-B-Q at 1,295 cm⁻¹, C–N stretching in B-B-B at 1,224 cm⁻¹, aromatic C−H in-plane bending vibration at 1,144 cm⁻¹, and aromatic C–H out of plane bending vibration of 1,2,4-ring at 806 cm⁻¹ (19, 20).

Preparation of Ink and Coating.

The PANI dispersion was obtained by mixing 30 mg of PANI-EB powder and 1.5 mL of D.I. water and sonicating for 1 h. Sonication treatment was processed to redisperse polyanilines into the original nanoscale fibers. The fluorous surfactant solution was prepared by dissolving 100 mg of KPD in 2 mL of hexafluoroisopropanol, which is miscible with water. The fluorous PANI (F-PANI) dispersion was obtained by sonicating 600 µL of polymer dispersion and 100 µL of fluorous surfactant solution for 30 min. In this process, the noncovalent interactions between KPD (amide and ether groups) and the imines of the PANI nanofibers are introduced.

Polymer coatings were prepared by fabricating PANI inks onto the substrates (NC membrane or filter paper). Test line bands with an area of 45 mm² (1.5 cm * 0.3 cm) were created using 17 µL of ink, while bands measuring 105 mm² (1.5 cm * 0.7 cm) and 210 mm² (1.5 cm * 1.4 cm) were created using 40 µL and 80 µL of ink, respectively. The thickness of the test line bands was obtained by the stylus profilometer, and the average value was 27.9 ± 5.9 µm (n = 5). We used the test lines for measurement after drying for 2 h.

Calculation of Conductivity of the Coating.

We calculated the conductivity by using the four-point probe method. We recorded the resistance reading (R) and a constant (C = 4.3947) derived from the dimensions of our sample (24), and determined the sheet resistance (ρ_s) using the following equation:

$${\rho }_s=R\ast C,$$

$$\sigma =\frac{1}{{\rho }_s\ast l}.$$

The resistivity of the coating is ρ_s multiplied by its thickness (l), and the conductivity (σ) is its reciprocal. The resistance (R) values were collected from over 30 different locations with three samples for each concentration.

Supplementary Material

Appendix 01 (PDF)

Data, Materials, and Software Availability

SI Appendix data have been deposited in PNAS. All other study data are included in the article and/or SI Appendix.