Biosensors for Selective and Label-free Detection of Xylella fastidiosa

The Xylella fastidiosa bacterium is one of the most hostile pathogenic microorganisms causing multiple plant diseases, with huge economic impact on both agriculture and environment. By revealing the presence of the bacterium before symptom arrival, preventive action plans could be applied to confine the contagion. In this scenario, the development of a surveillance device, conveying high sensitivity along with early detection of X. fastidiosa outbreaks, would be of paramount importance. Fundamental to the development of such a detection system is the availability of high binding affinity antibodies for X. fastidiosa (anti-XF), which could be integrated in the sensor transducers. Also, a label-free detection system would be desirable, which shorts the analysis process to achieve optimal time-to-results for pathogen identification.
In this study Surface Plasmon Resonance (SPR) has been used to assess an optimized biofunctionalization protocol of gold surfaces with anti-XF, validating their capturing efficacy against X. fastidiosa. Among other bio-sensing techniques, SPR holds the advantage of being a label-free detection system, providing real-time monitoring of bio-affinity reactions. So far, a variety of plant pathogen biomarkers were studied by means of SPR, but none of them involves X. fastidiosa.
The bio-functionalization of gold transducing interfaces was performed with the polyclonal antibodies for X. fastidiosa, covalently bounded to a self-assembled monolayer of alkylthiols. This configuration guaranteed the assay selectivity, which is assessed by means of a control experiment with the non-binding Burkholderia phytofirmans bacterium. The SPR sensogram of the binding X. fastidiosa is depicted in Figure 1A, and the comparison with the control experiment response is reported in Figure 1B.
Remarkably, a limit of detection as low as 10⁵ CFU/mL was achieved by transducing the direct interaction between the X. fastidiosa bacterium and its affinity antibody, which is comparable to the label-needing ELISA gold standard. Moreover, the binding-affinity between polyclonal antibodies and the X. fastidiosa bacteria has been also evaluated, obtaining an equilibrium affinity constant of 3.5∙10⁷ M-1, comparable with those given in the literature for bacteria detection against affinity antibodies. The study is therefore a preliminary development of a reliable cost-effective process to successfully bio-functionalize a gold surface, eventually suitable as gate electrode in wide-field bioelectronic sensors, for ultra-sensitive detection of X. fastidiosa.