Vertical flow-based paper immunosensor for rapid electrochemical and colorimetric detection of influenza virus using a different pore size sample pad

Abstract

We present a novel vertical flow-based paper immunosensor for the rapid and sensitive electrochemical and colorimetric detection of influenza H1N1 viruses using a different pore size (DP) sample pad. The DP sample pad consisted of two different pore size papers: larger pores (diameter: 11 mu m) facing the inlet, and smaller pores (diameter: 0.45 mu m) facing the conjugate pad. This sample pad offered moderate and uniform flows, and hence concentrated horseradish peroxidase tagged antibodies (HRP-Abs)-H1N1 complexes from 40 mu L of sample volumes on a conjugate pad for 2 min after sample injection, thereby providing fast detection (6 min for both detection methods) with 100 mu L of flushing afterwards, high sensitivity, and the simplicity of the sensor. The filtration characteristics of the DP sample pad were evaluated using fluorescent beads, indicating that only small sized bio-particles such as viruses can pass through the sample pad. Sandwich immunoreactions of HRP-Ab-H1N1-Ab were performed on the gold paper electrode of the immunoStrip, which was determined by electrochemical impedance spectroscopy (EIS) measurements. Simultaneously, the color signal of free HRP-Ab captured on the colorimetric zone was obtained using a scanner, and the intensity was analyzed using ImageJ. This immunosensor detected H1N1 virus concentration as low as 3.3 plaque forming units (PFU)/mL (phosphate buffer saline; PBS) and 4.7 PFU/mL (saliva) by EIS, and 1.34 PFU/mL (PBS) and 2.27 PFU/mL (saliva) by the colorimetric method. Furthermore, integrating these two detection methods can reduce false results with double assurance, and this device can provide a simple and economical on-site detection platform.