Surface Plasmon Resonance Assay for Label‐Free and Selective Detection of Xylella Fastidiosa

Abstract

Xylella fastidiosa is among the most dangerous plant bacteria worldwide causing a variety of diseases, with huge economic impact on agriculture and environment. A surveillance tool, ensuring the highest possible sensitivity enabling the early detection of X. fastidiosa outbreaks, would be of paramount importance. So far, a variety of plant pathogen biomarkers are studied by means of surface plasmon resonance (SPR). Herein, multiparameter SPR (MP‐SPR) is used for the first time to develop a reliable and label‐free detection method for X. fastidiosa. The real‐time monitoring of the bioaffinity reactions is provided as well. Selectivity is guaranteed by biofunctionalizing the gold transducing interface with polyclonal antibodies for X. fastidiosa and it is assessed by means of a negative control experiment involving the nonbinding Paraburkholderia phytofirmans bacterium strain PsJN. Limit of detection of 105 CFU mL−1 is achieved by transducing the direct interaction between the bacterium and its affinity antibody. Moreover, the binding affinity between polyclonal antibodies and X. fastidiosa bacteria is also evaluated, returning an affinity constant of 3.5 × 107 m−1, comparable with those given in the literature for bacteria detection against affinity antibodies.

Fast and Reliable Electronic Assay of a Xylella fastidiosa Single Bacterium in Infected Plants Sap

“…Gate Biofunctionalization Protocol: The sensing gate electrode was biofunctionalized following a protocol published elsewhere. [25,21,32,34] In the following the salient information is given. The biofunctionalization started with a 3-MPA and 11-MUA mixed chemical self-assembled monolayer (SAM) activated with EDC-sulfoNHS chemistry.…”

“…A multiparameter surface plasmon resonance study proved that this protocol enabled to reach a very highly dense coverage of capturing anti-Xf of 3.6 ± 0.8 × 10 11 molecules cm −2 . [25] Diffusion of a Single Bacterium at a Millimeter-Wide Capturing Surface: The free diffusion constant of a bacterium in a bulk liquid is D = 200 − 1900 μm 2 s −1 . [27,35] This implied, according to Einstein equation Δr = (6 D × Δt) 1/2 , that in a Δt of 600s, the bacterium spanned on average a sphere with a radius Δr of about 0.8-2.6 mm.…”

“…A multiparameter surface plasmon resonance study proved that this protocol enabled to reach a very highly dense coverage of capturing anti ‐Xf of 3.6 ± 0.8 × 10 11 molecules cm −2 . [ 25 ]…”

“…[ 7 ] Lately also, a surface‐plasmon resonance label‐free approach has been successfully undertaken. [ 25 ] These approaches are based on bench‐top instrumentations, hardly prone for fast and convenient in‐field detections. Moreover, while qPCR‐based techniques can reach limit‐of‐quantifications (LOQs) of 10 2 colony‐forming‐unit (cfu) per mL, [ 24 ] all the other assays can, at the very best, detect 10 4 –10 5 cfu mL −1 .…”

Hemolysis-Inspired, Highly Sensitive, Label-Free IgM Detection Using Erythrocyte Membrane-Functionalized Nanomechanical Resonators

“…However, these labeling methods cause false-positive/negative occurrences in biological assays due to alterations in physicochemical or intermolecular binding properties [ 22 , 23 ]. To solve this problem, label-free methods for biomolecular detection have been developed, such as electrochemical [ 24 , 25 , 26 ], surface plasmon resonance [ 27 , 28 ], and nanowell assay [ 29 , 30 , 31 ]. The label-free immunoassay methods are attracting attention because of their advantages, such as easy fabrication [ 32 ] and low cost [ 33 ], while excluding the effect of the labels [ 34 ].…”