Ionic Liquid Aqueous Two-Phase Systems for the Enhanced Paper-Based Detection of Transferrin and Escherichia coli

Abstract

Aqueous two-phase systems (ATPSs) have been widely utilized for liquid-liquid extraction and purification of biomolecules, with some studies also demonstrating their capacity as a biomarker concentration technique for use in diagnostic settings. As the limited polarity range of conventional polymer-based ATPSs can restrict their use, ionic liquid (IL)-based ATPSs have been recently proposed as a promising alternative to polymer-based ATPSs, since ILs are regarded as tunable solvents with excellent solvation capabilities for a variety of natural compounds and proteins. This study demonstrates the first application of IL ATPSs to point-of-care diagnostics. ATPSs consisting of 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim][BF₄]) and sodium phosphate salt were utilized to quickly concentrate biomarkers prior to detection using the lateral-flow immunoassay (LFA). We found the phase separation speed of the IL ATPS to be very rapid and a significant improvement upon the separation speed of both polymer-salt and micellar ATPSs. This system was successfully applied to both sandwich and competitive LFA formats and enhanced the detection of both Escherichia coli bacteria and the transferrin protein up to 8- and 20-fold, respectively. This system's compatibility with a broad range of biomolecules, rapid phase separation speed, and tunability suggest wide applicability for a large range of different antigens and biomarkers.

Keywords: Escherichia coli; aqueous two-phase systems; ionic liquid; lateral-flow immunoassay; transferrin.

Figure 1

Schematic of biomarker concentration with an IL ATPS and subsequent detection on the LFA. Gold nanoprobes and phase-forming components are first mixed with a known concentration of target analyte. Following phase separation, the top phase is extracted and applied to both the competitive format LFAs for detection of transferrin and sandwich format LFAs for the detection of E. coli. For the competitive format LFA, which utilizes purple-colored DGNPs, a positive test is indicated by a single purple band, while a negative test is indicated by two purple bands. For the sandwich format LFA, which utilizes red-colored GNPs, a positive test is indicated by two red bands, while a negative test is indicated by one red band.

Figure 2

Visualization of phase separation and speed of an IL ATPS. Purple-colored dextran-coated gold nanoparticles partitioned extremely to the top phase and were used to visualize separation. ATPSs separated within (A) 1 min for the 1:1 ATPS, and (B) 5 min for the 1:9 ATPS.

Figure 3

Limit of detection for LFA tests detecting for protein transferrin. (A) Detection limit for the LFA-only test was found to be 5 ng/μL. (B) Detection limit for the LFA when combined with a 1:1 ATPS was 1.25 ng/μL, and (C) detection limit for the LFA when combined with a 1:9 ATPS was 0.25 ng/μL, indicating 4-fold and 20-fold improvements, respectively.

Figure 4

MATLAB analysis of transferrin LFA tests. Data for (▴) LFA-only tests without ATPS enhancement, (■) enhancement with a 1:1 ATPS, and (●) enhancement with a 1:9 ATPS. At each concentration, test line intensities in arbitrary units (a.u.) were lower for the 1:1 ATPS/LFA test than the LFA-only test and lower for the 1:9 ATPS/LFA test than the 1:1 ATPS/LFA test, indicating improved detection with more extreme volume ratios.

Figure 5

Limit of detection for LFA tests detecting for Escherichia coli. (A) Detection limit for the LFA-only test was found to be 3.6 × 10⁵ cfu/mL. (B) Detection limit for the LFA when combined with a 1:1 ATPS was 1.8 × 10⁵ cfu/mL and (C) detection limit for the LFA when combined with a 1:9 ATPS was 4.5 × 10⁴ cfu/mL, indicating 2-fold and 8-fold improvements, respectively.

Figure 6

MATLAB analysis of E. coli LFA tests. Data for (▴) LFA-only tests without ATPS enhancement, (■) enhancement with a 1:1 ATPS, and (●) enhancement with a 1:9 ATPS. At each concentration, test line intensities in arbitrary units (a.u.) were greater for the 1:1 ATPS/LFA test than the LFA-only test and greater for the 1:9 ATPS/LFA test than the 1:1 ATPS/LFA test, indicating improved detection with more extreme volume ratios.