Phage-displayed antibody fragments in microfluidic paper-based devices: a novel approach for sensitive detection of glycine-extended gastrin 17 biomarker using gold nanoparticles

Abstract

To evaluate the potential use of phage-displayed recombinant antibody fragments as biorecognition elements on microfluidic paper-based devices (µPADs), phage-displayed VL and soluble VL antibody fragments were immobilized on the chitosan-modified surface of µPADs to detect glycine-extended gastrin 17 (G17-Gly) an integral peptide biomarker for colorectal cancer. Additionally, the phage shaft displaying the scFv antibody fragment, used as a detection probe, was conjugated with gold nanoparticles (GNPs) and characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), and UV–visible spectroscopy (UV–Vis). Following the microfluidic sandwich immunoassay, the mean intensity of the color spots was quantitatively analyzed using an image analysis program. Peptide calibration curves showed a linear relationship between the intensity of the color spot signal and the logarithm of the peptide concentration within the ranges of 10⁻⁶–5 × 10⁻¹ µM (R² = 0.98) for the phage-VL fragment and 10⁻⁴–1 µM (R² = 0.97) for the soluble VL fragment, with limits of detection (LOD) of 0.9 and 29 pM, respectively. The proposed µPAD-based immunoassay with the desirable LODs without further amplification provides a simple, versatile means for detecting biomarkers and pathogens of interest.