An enzymatic nucleic acid vertical flow assay

Area of Science:

• Biotechnology
• Analytical Chemistry
• Molecular Diagnostics

Background:

• Lateral flow assays have limitations in multiplexing, quantitation, and hook effect.
• Vertical flow assays offer potential improvements over traditional lateral flow methods.
• Paper-based assays provide a low-cost, accessible platform for diagnostics.

Purpose of the Study:

• To develop the first passive paper-based vertical flow assay for nucleic acid target detection.
• To optimize assay parameters for enhanced signal-to-noise ratio.
• To investigate the use of an enzyme reporter for signal amplification in a vertical flow assay.

Main Methods:

• Development of a passive paper-based vertical flow assay.
• Utilized horseradish peroxidase as an enzymatic tracer for signal amplification.
• Optimized assay parameters including sample preparation (heat denaturation, probe hybridization) and assay format (sandwich assay).

Main Results:

• Achieved a limit of detection (LOD) of 1.7 nM (8.5 fmol dsDNA in 12 µl).
• Established a linear range of 3-194 nM with a high correlation coefficient (0.978).
• Demonstrated 100% consistency with real-time PCR for hepatitis B virus detection in 45 clinical plasma samples.

Conclusions:

• The developed passive paper-based vertical flow assay is a sensitive and specific method for nucleic acid detection.
• The assay offers rapid analysis (6 minutes) with a small sample volume (12 µl).
• This enzyme-reporter-based vertical flow assay shows high potential for clinical diagnostics and point-of-care applications.