Fully Dried Two-Dimensional Paper Network for Enzymatically Enhanced Detection of Nucleic Acid Amplicons

Area of Science:

• Biomedical Engineering
• Analytical Chemistry
• Diagnostics

Background:

• Paper-based analytical devices (PADs) offer potential for point-of-care diagnostics.
• Current PADs for immunoassays often require manual addition of reagents and are limited to protein targets.

Purpose of the Study:

• To develop a novel two-dimensional paper network (2DPN) device for enhanced nucleic acid detection.
• To overcome limitations of existing PADs by integrating dried signal enhancement reagents and enabling detection of nucleic acid targets.

Main Methods:

• Device design guided by mathematical equations for optimal fluidic control.
• Integration of dried solutions of hydrogen peroxide, diaminobenzidine, and horseradish peroxidase within the 2DPN.
• Evaluation of limit-of-detection (LOD), stability, and detection versatility for various nucleic acid types.

Main Results:

• Achieved a limit-of-detection of 5 × 10^11 nucleic acid copies/mL without increased false positives.
• Device demonstrated stability for 28 days under dry storage.
• Reliable signal enhancement observed within 40 minutes of sample application.
• Successfully detected double-stranded DNA, single-stranded DNA, and peptide nucleic acids.

Conclusions:

• The novel 2DPN device offers an easy-to-use, stable, and sensitive platform for nucleic acid detection.
• Integrated dried reagents and optimized fluidics enhance assay performance for point-of-care applications.
• The versatile detection capabilities support a broad range of diagnostic applications.