title: 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.
authors.affiliation: Department of Bioengineering, University of California, Los Angeles, Los Angeles, California, United States of America.
authors.forename: Ricky Y T
authors.initials: RY
authors.lastname: Chiu
authors.affiliation: Department of Bioengineering, University of California, Los Angeles, Los Angeles, California, United States of America.
authors.forename: Alison V
authors.initials: AV
authors.lastname: Thach
authors.affiliation: Department of Bioengineering, University of California, Los Angeles, Los Angeles, California, United States of America.
authors.forename: Chloe M
authors.initials: CM
authors.lastname: Wu
authors.affiliation: Department of Bioengineering, University of California, Los Angeles, Los Angeles, California, United States of America.|Division of Advanced Prosthodontics & Weintraub Center for Reconstructive Biotechnology, UCLA School of Dentistry, Los Angeles, California, United States of America.
authors.forename: Benjamin M
authors.initials: BM
authors.lastname: Wu
authors.affiliation: Department of Bioengineering, University of California, Los Angeles, Los Angeles, California, United States of America.
authors.forename: Daniel T
authors.initials: DT
authors.lastname: Kamei
mesh_terms: D000818:Animals; D002417:Cattle; D007118:Immunoassay / Q000379:methods*; D007159:Immunologic Tests; D057230:Limit of Detection; D053758:Nanoparticles; D000067716:Point-of-Care Testing; D012680:Sensitivity and Specificity