An Aqueous Two-Phase System for the Concentration and Extraction of Proteins from the Interface for Detection Using the Lateral-Flow Immunoassay

Abstract

The paper-based immunoassay for point-of-care diagnostics is widely used due to its low cost and portability over traditional lab-based assays. Lateral-flow immunoassay (LFA) is the most well-established paper-based assay since it is rapid and easy to use. However, the disadvantage of LFA is its lack of sensitivity in some cases where a large sample volume is required, limiting its use as a diagnostic tool. To improve the sensitivity of LFA, we previously reported on the concentration of analytes into one of the two bulk phases of an aqueous two-phase system (ATPS) prior to detection. In this study, we preserved the advantages of LFA while significantly improving upon our previous proof-of-concept studies by employing a novel approach of concentrating gold nanoparticles, a common LFA colorimetric indicator. By conjugating specific antibodies and polymers to the surfaces of the particles, these gold nanoprobes (GNPs) were able to capture target proteins in the sample and subsequently be concentrated within 10 min at the interface of an ATPS solution comprised of polyethylene glycol, potassium phosphate, and phosphate-buffered saline. These GNPs were then extracted and applied directly to LFA. By combining this prior ATPS interface extraction with LFA, the detection limit of LFA for a model protein was improved by 100-fold from 1 ng/μL to 0.01 ng/μL. Additionally, we examined the behavior of the ATPS system in fetal bovine serum and synthetic urine to more closely approach real-world applications. Despite using more complex matrices, ATPS interface extraction still improved the detection limit by 100-fold within 15 to 25 min, demonstrating the system’s potential to be applied to patient samples.

Sensitivity enhancement in lateral flow assays: a systems perspective

“…polymers, salts, or micelles) at high concentration to form two immiscible aqueous phases, allowing concentration of a target protein in one phase. 23 While ATPS were demonstrated to work as a preconcentration step before the application of sample to a bacteriophage M13 24 and a transferrin 25 LFA, Pereira et al integrated the APTS step into a "three-dimensional" design to improve sensitivity 10-fold in a Plasmodium lactate dehydrogenase (pLDH) LFA. 26…”

Paper-Based Biosensors: Frontiers in Point-of-Care Detection of COVID-19 Disease

“…Several methods to improve LFA sensitivity have been used, such as enrichment or concentration methods, divided into on-strip and off-strip pre-enrichment, temperature-humidity control, enzyme-based signal enhancement, etc. [58][59][60][61]. However, research on simplified and lowcost methods is still needed.…”

Ways to Reach Lower Detection Limits of Lateral Flow Immunoassays

“…Similar solutions are offered by other manufacturers. A special aqueous two-phase system for the concentration of protein analytes, containing polyethylene glycol, potassium phosphate, and phosphate-buffered saline, was used by Chiu et al [10]. With its help, a 100fold reduction in the detection limit was achieved.…”