DNA-only, microwell-based bioassay for multiplex nucleic acid detection with single base-pair resolution using...

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Biochemistry

Background:

• Nucleic acid detection is crucial for disease diagnostics, but current methods face challenges like amplification errors and enzyme instability.
• Existing techniques often require target amplification or protein-based signal amplification, limiting their accuracy and stability.

Purpose of the Study:

• To develop a novel, amplification-free system for sensitive nucleic acid detection using multicomponent nucleic acid enzymes (MNAzymes).
• To demonstrate singleplex and multiplex detection capabilities of MNAzymes in various formats, including microwells.
• To establish a protein-free diagnostic platform with high sensitivity and single base-pair resolution.

Main Methods:

• Development and validation of multicomponent nucleic acid enzymes (MNAzymes) for nucleic acid detection.
• Testing MNAzyme performance in solution, on beads, and in femtoliter-sized microwells.
• Demonstration of multiplexed detection using MNAzymes in buffer and clinical samples (nasopharyngeal swabs).

Main Results:

• Achieved femtomolar (fM) detection limits for nucleic acid targets without amplification or protein enzymes.
• Demonstrated highly sensitive (180 fM limit) and specific detection in femtoliter microwells, surpassing existing NAzyme-mediated methods.
• Successfully performed multiplexed nucleic acid detection in nasopharyngeal swab samples with superior single base-pair resolution.

Conclusions:

• MNAzymes offer a versatile, protein-free platform for sensitive and specific nucleic acid detection.
• The developed microwell-based system represents a significant advancement in diagnostic sensitivity and simplicity.
• This technology holds potential for next-generation diagnostic tools, including digital assays with single-molecule resolution.