Quantification of avian influenza A virus (H5N1) via enzymatic reactions and glucometer test strips

Abstract

A sensor capable of quantifying avian influenza A virus (subtype H5) was developed by targeting hemagglutinin (HA) protein. This was accomplished by designing a solution-based sandwich assay that generated orthophosphate (PO₄³⁻) in proportion to the concentration of HA protein (and hence virus particles) present in a sample. Virus particles were captured and concentrated using an H5-specific primary antibody conjugated to magnetic microparticles. Following magnetic separation, an alkaline phosphatase (AP)-conjugated secondary antibody was introduced to bind to the captured virus. The resulting assembly was exposed to p-nitrophenyl phosphate (PNPP), producing PO₄³⁻ in proportion to the amount of virus present. The generated phosphate was subsequently converted to glucose through the addition of maltose and maltose phosphorylase, and the resulting glucose was quantified amperometrically using an off-the-shelf commercial glucose test strip interfaced with a mini-potentiostat. Using this approach, limits of detection of 7.78 × 10⁵ RNA copies per mL in binding buffer, 1.46 × 10⁶ RNA copies per mL in artificial saliva, and 1.26 × 10⁶ RNA copies per mL in tap water were obtained. Selectivity was demonstrated against respiratory pathogen pools, representative Gram-positive and Gram-negative bacteria, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This work presents a modular immunoassay-based strategy that leverages widely available glucose test strips for quantitative viral detection in a laboratory proof-of-concept format and may be adaptable to other targets through substitution of capture and detection antibodies.