Enhancing the Sensitivity of DNA and Aptamer Probes in the Dextran/PEG Aqueous Two-Phase System

Abstract

Increasing the local concentration of DNA-based probes is a convenient way to improve the sensitivity of biosensors. Instead of using organic solvents or ionic liquids that phase-separate with water based on hydrophobic interactions, we herein studied a classic aqueous two-phase system (ATPS) comprising polyethylene glycol (PEG) and dextran. Polymers of higher molecular weights and higher concentrations favored phase separation. DNA oligonucleotides are selectively enriched in the dextran-rich phase unless the pH was increased to 12. A higher volume ratio of PEG-to-dextran and a higher concentration of PEG also enrich more DNA probes in the dextran-rich phase. The partition efficiency of the T15 DNA was enriched around seven times in the dextran phase when the volume ratio of dextran and PEG reached 1:10. The detection of limit improved by 3.6-fold in a molecular beacon-based DNA detection system with the ATPS. The ATPS also increased the sensitivity for the detection of Hg2+ and adenosine triphosphate, although these target molecules alone distributed equally in the two phases. This work demonstrates a simple method using water soluble polymers to improve biosensors.

β-Cyclodextrin Polymer-Based Host–Guest Interaction and Fluorescence Enhancement of Pyrene for Sensitive Isocarbophos Detection

“…At present, aptamers have been widely used as artificial antibodies in fluorescence methods because of their high affinity, excellent stability, and prolonged storage life. 18 , 19 For example, Dou et al have established a gold-based nanobeacon fluorescence probe for organophosphorus pesticide sensing, with a detection limit of quantification reaching 10 μg/L. 16 In order to monitor trace-level organophosphorus pesticide residuals and overcome the current issue of severe pollution, it is urgent to develop highly sensitive fluorescence methods.…”

Microfluidic Control of Coexisting Chemical Microenvironments within Multiphase Water-in-Fluorocarbon Droplets

“…3,26,31−35 Solute localization within distinct coexisting aqueous phases, both as a bulk system and as droplets, has been extensively studied for use in applications such as biomolecular purification and extraction, 24,36−40 bioengineering, 28,41−43 modeling of membraneless organelles, 11,44−48 and reactant localization. [12][13][14]49 Such phase-separated systems have also been studied in the form of emulsions, recently in conjunction with microfluidic technologies, resulting in the production of both all-aqueous 18,19,21,50−53 and water-inoil 54−57 emulsions with controllable morphologies, surface chemistry, and encapsulated components. However, while TLL has long been used as a means of controlling local solute concentration within bulk ATPS, 24,27,32,34,38,39 such efforts have generally not been undertaken for droplet-based systems, due to the difficulty in selecting specific phase compositions on an individual droplet level.…”

“…The physical chemistry governing bulk aqueous phase separation has been explored for decades, particularly as a means of using neutral polymer or polymer/salt aqueous two phase systems (ATPS) in biological separations, as included solutes typically demonstrate preferential partitioning among the phases present. − Given an arbitrary two phase system, a phase diagram maps the total polymer compositions that result in either single phase or phase-separated solutions. For any composition within the phase-separated region, there exists a tie line connecting it to two points on the binodal line, which give the polymer composition of the individual phases (Figure B). ,,, The tie line length (TLL) reflects the disparity in composition between the two phases; longer tie lines indicate more distinct phases, and lead to stronger solute partitioning. ,,− Solute localization within distinct coexisting aqueous phases, both as a bulk system and as droplets, has been extensively studied for use in applications such as biomolecular purification and extraction, ,− bioengineering, ,− modeling of membraneless organelles, ,− and reactant localization. − , Such phase-separated systems have also been studied in the form of emulsions, recently in conjunction with microfluidic technologies, resulting in the production of both all-aqueous ,,,− and water-in-oil − emulsions with controllable morphologies, surface chemistry, and encapsulated components. However, while TLL has long been used as a means of controlling local solute concentration within bulk ATPS, ,,,,, such efforts have generally not been undertaken for droplet-based systems, due to the difficulty in selecting specific phase compositions on an individual droplet level.…”

Ultrafast Ratiometric Detection of Aflatoxin B1 Based on Fluorescent β-CD@Cu Nanoparticles and Pt2+ Ions

“…Alternatively, various antibody-based immunoassay systems have been developed such as enzyme-linked immunosorbent assays and lateral flow strips. , These methods provide rapid detection for mycotoxins and enable on-site detection. However, long period in antibody preparation and high interbatch variations significantly impede its applications. , As a potential substitute for antibodies, nucleic acid aptamer has easy preparation and mass production advantages . However, the solution medium easily disturbs the aptamer recognition to target, which hinders their commercial application of aptamer-based sensors for AFB1 detection. , Thus, it is still challenging to achieve rapid recognition, high sensitivity, and low-cost detection of AFB1.…”

“…10,11 As a potential substitute for antibodies, nucleic acid aptamer has easy preparation and mass production advantages. 12 solution medium easily disturbs the aptamer recognition to target, which hinders their commercial application of aptamerbased sensors for AFB1 detection. 11,13 Thus, it is still challenging to achieve rapid recognition, high sensitivity, and low-cost detection of AFB1.…”