MED: Multiplexed, Entropy-Driven Catalysis, Single-Nucleotide-Resolved ctDNA Detection

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Nanotechnology

Background:

• Real-time monitoring of circulating tumor DNA (ctDNA) is crucial for early cancer detection, treatment response assessment, and identifying resistance.
• Existing ctDNA detection methods often lack the scalability and accessibility required for point-of-care applications.
• There is a significant need for sensitive assays capable of resolving single-nucleotide variations in ctDNA.

Purpose of the Study:

• To develop a multiplexed, single-nucleotide-resolved assay for the detection of ctDNA.
• To enable enzyme-free nucleic acid amplification and visual detection of ctDNA at the point-of-care.
• To assess the diagnostic accuracy and clinical potential of the developed assay.

Main Methods:

• Introduction of a multiplexed, entropy-driven catalysis (EDC) assay for single-nucleotide-resolved ctDNA detection.
• Utilizing a triple amplification strategy involving EDC, silver nanoparticle (AgNP) catalyzed Ag+ release, and urease-catalyzed urea hydrolysis for colorimetric readout.
• Integration with bioactive paper and smartphone readout for point-of-care application.

Main Results:

• The MED assay demonstrated 100% concordance with sequencing for detecting a panel of four ctDNAs in 40 blood specimens.
• The assay achieved over 85% accuracy in distinguishing between cancer and non-cancer samples.
• Integrated point-of-care format provided rapid (within 45 min) and accessible results with over 70% accuracy.

Conclusions:

• The MED assay offers a scalable, enzyme-free, and single-nucleotide-resolved method for ctDNA detection.
• The point-of-care format provides rapid, multiplexed, and accessible results with significant clinical potential.
• This technology can enhance early cancer diagnosis, dynamic monitoring, and personalized therapeutic strategies.