On-Chip Enrichment System for Digital Bioassay Based on Aqueous Two-Phase System

Area of Science:

• Biotechnology
• Analytical Chemistry
• Molecular Diagnostics

Background:

• Digital bioassays require high sensitivity for accurate molecular detection.
• On-chip enrichment methods can improve assay performance but often need complex instrumentation.
• Aqueous two-phase systems (ATPS) offer a promising platform for molecular separation and concentration.

Purpose of the Study:

• To develop and evaluate an on-chip enrichment method using dextran/polyethylene glycol aqueous two-phase systems (DEX/PEG ATPS) for digital bioassays.
• To demonstrate the system's effectiveness in enriching various biomolecules, including DNA, RNA, and proteins.
• To assess the potential of this technology for sensitive, portable diagnostic applications.

Main Methods:

• Fabrication of an array device with millions of femtoliter reactors.
• Utilizing DEX-rich droplets within a PEG-rich medium for molecular enrichment.
• Genetically tagging alkaline phosphatase (ALP) with a DEX-binding domain (DBD) for enrichment.
• Performing Cas13-based digital SARS-CoV-2 RNA detection assays.
• Enrichment experiments with non-tagged β-galactosidase using DBD-tagged antibodies.

Main Results:

• Achieved a 59-fold enrichment of genetically tagged ALP molecules in DEX droplets.
• Demonstrated a 31-fold enrichment for target RNA molecules in a Cas13-based SARS-CoV-2 assay.
• Obtained over 100-fold enrichment for non-tagged β-galactosidase using DBD-tagged antibodies.
• Confirmed effective on-chip enrichment of target molecules without external instruments or power.

Conclusions:

• The developed DEX/PEG ATPS on-chip system enables efficient molecular enrichment for digital bioassays.
• This technology significantly enhances detection sensitivity, applicable to DNA, RNA, and protein analysis.
• The system's portability and lack of external power requirements make it suitable for on-site diagnostics and portable testing devices.