Partitioning-Induced Isolation of Analyte and Analysis via Multiscaled Aqueous Two-Phase System

Abstract

Most fluorescence-based bioanalytical applications need labeling of analytes. Conventional labeling requires washing to remove the excess fluorescent labels and reduce the noise signals. These pretreatments are labor intensive and need specialized equipment, hindering portable applications in resource-limited areas. Herein, we use the aqueous two-phase system (ATPS) to realize the partitioning-induced isolation of labeled analytes from background signals without extra processing steps. ATPS is formed by mixing two polymers at sufficiently high concentrations. ATPS-based isolation is driven by intrinsic affinity differences between analytes and excess labels. To demonstrate the partitioning-induced isolation and analysis, fluorescein isothiocyanate (FITC) is selected as the interfering fluorophore, and a monoclonal antibody (IgG) is used as the analyte. To optimize ATPS compositions, different molecular weights and mass fractions of polyethylene glycol (PEG) and dextran and different phosphate-buffered saline (PBS) concentrations are investigated. Various operational scales of our approach are demonstrated, suggesting its compatibility with various bioanalytical applications. In centimeter-scale ATPS, the optimized distribution ratios of IgG and FITC are 91.682 and 0.998 using PEG 6000 Da and dextran 10,000 Da in 10 mM PBS. In millimeter-scale ATPS, the analyte is enriched to 6.067 fold using 15 wt % PEG 35,000 Da and 5 wt % dextran 500,000 Da in 10 mM PBS. In microscale ATPS, analyte dilutions are isolated into picoliter droplets, and the measured fluorescence intensities linearly correlated with the analyte concentrations (R 2 = 0.982).

Self-lubricating drops

“…However, since the design options are so huge, constructing a design frame is necessary. (4) Broad applications from complex composition: Aqueous two-phase systems, known as the clean alternatives for traditional organic-water solvent extraction, make self-lubricating drops suitable for bioengineering processes or environment managements [34,98,99]. It is also notable that liquid crystalline solutions can broaden the applied range for fabrication of semiconductors, photo-alignment [6,95,100,101],…”

Liquid-Liquid Phase Separation for Analyte Enrichment in Coacervates for Ultra-Sensitive Biosensing

“…This segregation increases local concentrations, thus bolstering detection sensitivity. [16][17][18][19] It was reported that DNA could be enriched around seven times in ATPS and its detection of limit could be improved by 3.6-fold. 19 In living cells, a large variety of membraneless organelles are biomolecular condensates providing crowded compartments for biomolecules to fulfill their biochemical functions.…”