Micro-Probe Potentiometric pH Sensor for Detection of Amplification in the LAMP Assay for White Spot Syndrome Virus (WSSV) in Shrimps

A novel application of pH sensors is in the detection of amplification in nucleic acid-based methods for diagnosis of organisms. The detection is based on the production of hydrogen ions during the amplification process. In this study, a micro-probe potentiometric sensor for pH was fabricated and applied in the reagentless detection of the White Spot Shrimp Virus (WSSV) through the loop-mediated isothermal amplification (LAMP) technique. The pH micro-probe contained a pencil graphite modified with polypyrrole nanotubes through galvanostatic electrodeposition in the presence of methyl orange (MO). Electropolymerization parameters and potentiometric measurements parameters were optimized to obtain maximum sensitivity. The probe was characterized using Field Emission–Scanning Electron Microscopy (FE-SEM) and Fourier Transform-Infrared (FT-IR) Spectroscopy. The sensor showed a sub-Nernstian response (−45.5 mV/pH) at pH range 2–8 and LOD of
${5.19}\times {10}^{-{8}}$
M; with minimum hysteresis (
$\Delta $
m = ± 1.27 mV/pH), and low potential drift (±1.00 mV, pH 8). The probe was used to measure the pH of the microliter-volume reaction mixture at the start and at the end of the LAMP assay for WSSV. The probe detected pH change (
$\Delta $
pH = −0.675) that occurred during the LAMP assay. This behavior correlated well with the results of fluorescence detection of the LAMP assay kit using SYBR Safe® DNA gel stain. A high diagnostic sensitivity (93.8%) and specificity (100%) was observed. The fabricated potentiometric sensor showed promising result in monitoring positive LAMP reaction through measurement of pH. This provides a method to diagnose shrimp samples with WSSV that is rapid, easy-to-operate, and cost-effective.