Results of LFA for detecting Tf in FBS.

<p>(A) Images of test strips without (top panel) and with (bottom panel) the prior concentration step using the ATPS interface extraction step. 50 μL sample solutions were applied to each LFA test strip. (B) MATLAB quantification of test signal intensity where a value above a threshold of 0.25 corresponded to a negative test.</p

Transition electron microscopy (TEM) image of naked gold nanoparticles.

<p>Nanoparticles were suspended in filtered ultrapure water. Length of the scale bar corresponds to 40 nm. ImageJ analysis indicated the particle diameter to be 20.0 ± 3.0 nm (n = 275).</p

Surface modification of GNP to influence partitioning behavior in ATPS.

<p>Schematic of GNP and the functionality of each component.</p

Schematic representation of the integration of ATPS interface extraction with competition-based LFA and the interpretations of the positive and negative results.

<p>An ATPS solution was constructed and allowed to phase separate for 10 min in PBS and 25 min in FBS and synthetic urine in a glass tube prior to the extraction of 30 μL of the interface containing GNPs. The extracted sample was then applied to an LFA test strip and results were read after 10 min for the PBS system and after 25 min for the FBS and synthetic urine systems. The appearance of only the control line indicated a positive result while the appearance of both the control and test lines indicated a negative result.</p

Results of LFA for detecting Tf in synthetic urine.

<p>(A) Images of test strips without (top panel) and with (bottom panel) the prior concentration step using the ATPS interface extraction step. 50 μL sample solutions were applied to each LFA test strip. (B) MATLAB quantification of test signal intensity where a value above a threshold of 0.25 corresponded to a negative test.</p

Results of LFA for detecting Tf in PBS.

<p>(A) Images of test strips without (top panel) and with (bottom panel) the prior concentration step using the ATPS interface extraction step. 50 μL sample solutions were applied to each LFA test strip. (B) MATLAB quantification of test signal intensity where a value above a threshold of 0.25 corresponded to a negative test.</p

Demonstration of the partitioning behavior of GNPs in our PEG-salt ATPS.

<p>Various amounts of PEG were conjugated to the GNPs to manipulate their partitioning behavior: (A) Using a molar ratio of 5800:1 PEG:GNP during conjugation, the resulting GNPs partitioned preferentially into the PEG-rich top phase. (B) Using a molar ratio of 1200:1 PEG:GNP during conjugation, the GNPs partitioned into the PEG-poor bottom phase. (C) Using a molar ratio of 3500:1 PEG:GNP during conjugation, the resulting GNPs partitioned exclusively to the interface. For (A), (B), and (C), the red observed at the very top of the liquid-air interface was due to a reflection and not due to the presence of nanoprobes. Studies were performed in glass tubes 12 x 75 mm in size.</p

Summary of the technical innovation of engineering particles capable of partitioning to the interface of an ATPS to concentrate a target and the improvements in PBS relative to our previous proof-of-concept studies.

<p>Summary of the technical innovation of engineering particles capable of partitioning to the interface of an ATPS to concentrate a target and the improvements in PBS relative to our previous proof-of-concept studies.</p

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

<div><p>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.</p></div