Label-free and Sensitive Detection of Citrus Bark Cracking Viroid in Hop Using Ti3C2Tx MXene-modified Genosensor

Abstract

Since the discovery of Citrus bark cracking viroid (CBCVd) in hops in 2007, affected hop-growing countries such as Slovenia and Germany have been actively pursuing efficient and easily accessible diagnostic tools that could contribute to the early detection of CBCVd in the field. In the early stages of CBCVd infection, typical symptoms or subtle signs of spread may not be evident. Detection becomes feasible only when the plant begins to display symptoms. Unfortunately, there is currently no treatment available, which requires the removal of infected plants as the only viable solution. However, this approach leads to significant economic losses on a large scale.This work demonstrates the development and study of a sensitive, selective, and label-free impedimetric genosensor for the detection of CBCVd in total RNA hop samples. The genosensor is based on a supporting glassy carbon electrode modified with streptavidin-agarose beads, which serve as an effective immobilization layer for a biotinylated single-stranded DNA capture probe. The integration of a 2D-layered Ti₃C₂T_x MXene into the sensing architecture resulted in a significantly improved electroanalytical performance of the genosensor. Several fabrication and operational parameters were optimized, such as the streptavidin-agarose beads deposition time, capture probe immobilization time and concentration, and sample incubation time. The optimized genosensor exhibited a limit of detection of only 0.5 fg μL⁻¹ (5.5 fmol L^-1) in combination with a one-hour incubation with the denatured total RNA hop extract, thus eliminating the need for an additional and laborious amplification step.