Detection of the Plant Pathogen Pseudomonas Syringae pv. Lachrymans on Antibody-Modified Gold Electrodes by Electrochemical Impedance Spectroscopy

Abstract

The present work describes an impedimetric immunosensor for Pseudomonas syringae pv. lachrymans (Psl) detection. This pathogen infects many crop species causing considerable yield losses, thus fast and cheap detection method is in high demand. In the assay, the gold disc electrode was modified with 4-aminothiophenol (4-ATP), glutaraldehyde (GA), and anti-Psl antibodies, and free-sites were blocked with bovine serum albumin (BSA). Sensor development was characterized by cyclic voltammetry (CV) and antigen detection by electrochemical impedance spectroscopy (EIS) measurements. Seven analyzed strains of Psl were verified as positive by the reference method (PCR) and this immunoassay, proving sensor specificity. Label-free electrochemical detection was in the linear range 1 × 103–1.2 × 105 CFU/mL (colony-forming unit) with an R2 coefficient of 0.992 and a detection limit (LOD) of 337 CFU/mL. The sensor did not interfere with negative probes like buffers and other bacteria. The assay was proven to be fast (10 min detection) and easy in preparation. The advantage was the simplicity and availability of the verified analyte (whole bacteria) as the method does not require sample pretreatment (e.g., DNA isolation). EIS biosensing technique was chosen as one of the simplest and most sensitive with the least destructive influence on the probes compared to other electrochemical methods.

Advances in Plant Disease Detection and Monitoring: From Traditional Assays to In-Field Diagnostics

“…More recently, EIS sensors were used to detect up to seven strains of Pseudomonas syringae pv. lachrymans ( Psl ), which is mainly responsible for diseases of many cucurbit species, causing considerable yield losses [ 49 ] ( Figure 3 ). The detection was possible in the linear range 1 × 10 3 –1.2 × 10 5 CFU mL −1 , showing a sensitivity 30 times higher than the loop-mediated isothermal amplification (LAMP) and also the ability to detect Psl strains.…”

“… ( a ) Impedance changes obtained with Au/4-ATP/GA/anti-Psl/BSA electrodes, in the case of different concentrations of Psl; ( b ) linear relation between charge transfer resistance changes (ΔRct) and bacteria concentrations (on the right) (reproduced from [ 49 ], licensed under the Creative Commons Attribution). …”

Biosensor Technologies for Early Detection and Quantification of Plant Pathogens

“…The team detected Psl with a linear detection range 10 3 –1.2 × 10 5 CFU/ml ( R 2 = 0.992) and a detection limit of 337 CFU/ml. The assay was 30 times more sensitive than the conventional LAMP method and the detection time took 10 min ( Cebula et al, 2019 ). If we compare these two ligands used in EIS technique, DNA-based biosensors offer better advantages than antibody-based.…”

Point of Care Sensors for Infectious Pathogens

“…Derived sensors can be operated in faradaic or non-faradaic modes over a wide-frequency range, typically 100 kHz to 0.1 Hz, with modeled or fitted circuits enabling an assessment of the capacitive and/or resistive components. The interfacial impedance (often “charge transfer resistance”) or (related) capacitance elements are most useful, and their response to target recruitment at a receptor modified electrode interface has been widely applied to the detection of bacteria, viruses, and their genetic material with sensitivities comfortably good enough to be diagnostically relevant.…”