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An enzymatic nucleic acid vertical flow assay.

Mehdi Tahmasebi1, Taravat Bamdad2, Winnie Edith Svendsen3

  • 1Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box 14115-111, Tehran, Iran.

Analytical and Bioanalytical Chemistry
|March 30, 2022
PubMed
Summary

A novel passive paper-based vertical flow assay offers rapid and sensitive nucleic acid detection. This enzyme-based assay demonstrates high accuracy, achieving 100% consistency with real-time PCR for hepatitis B virus detection.

Keywords:
ChemiluminescenceHepatitis B virusHorseradish peroxidaseIn vitro diagnosticLateral flow assayVertical flow assay

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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.