QCM immunoassay for recombinant cysteine peptidase: A potential protein biomarker for diagnosis of citrus canker

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Abstract

Citrus canker is one of the most important agricultural citrus diseases worldwide. It is caused by Xanthomonas citri subsp. citri (Xcc) bacterium that infects leaves and the fruits produce a cysteine peptidase (CPXaC), which makes it a potential target for the development of effective and rapid detection methods for citrus canker. We report here the studies on the development of piezoelectric immunoassay for CPXaC using a polyclonal antibody against CPXaC (anti-CPXaC). Three different strategies for covalent immobilization of anti-CPXaC on gold surfaces were evaluated by monitoring the frequency (Δf) and energy dissipation (ΔD) variation in real time when 64.5×10-8 mol L-1 CPXaC was added. Anti-CPXaC immobilized with 11-mercaptoundecanoic acid (MUA) showed the best relation between the frequency and dissipation factor variation, and strong values for the kinetic and equilibrium binding constant were obtained. The immunosensor showed a detection limit of 13.0 nmol L-1 with excellent specificity, showing no response for different proteins that include another cysteine peptidase that is used as a target to detect Xylella fastidiosa bacterium, responsible for another important citrus disease. These results provide good perspectives for the use of CPXaC as a new biomarker for citrus canker. © 2012 Elsevier B.V.

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Afonso, A. S., Zanetti, B. F., Santiago, A. C., Henrique-Silva, F., Mattoso, L. H. C., & Faria, R. C. (2013). QCM immunoassay for recombinant cysteine peptidase: A potential protein biomarker for diagnosis of citrus canker. Talanta, 104, 193–197. https://doi.org/10.1016/j.talanta.2012.11.003

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