Attomolar electrochemical direct and sandwich immunoassays for the ultrasensitive detection of tomato brown rugose fruit virus
Abstract Tomato brown rugose fruit virus (TBRFV; Tobamovirus fructirugosum) is a highly virulent tobamovirus that has emerged as a major global threat to tomato and pepper crops over the past decade. Early and ultra-sensitive detection of TBRFV is critical for effective disease management and the mi...
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BMC
2025-07-01
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| Series: | Plant Methods |
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| Online Access: | https://doi.org/10.1186/s13007-025-01407-3 |
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| author | Negin Rezaei Ahmad Moshaii Mohammad Reza Safarnejad Reza H. Sajedi Mahsa Rahmanipour Masoud Shams-Bakhsh |
| author_facet | Negin Rezaei Ahmad Moshaii Mohammad Reza Safarnejad Reza H. Sajedi Mahsa Rahmanipour Masoud Shams-Bakhsh |
| author_sort | Negin Rezaei |
| collection | DOAJ |
| description | Abstract Tomato brown rugose fruit virus (TBRFV; Tobamovirus fructirugosum) is a highly virulent tobamovirus that has emerged as a major global threat to tomato and pepper crops over the past decade. Early and ultra-sensitive detection of TBRFV is critical for effective disease management and the mitigation of agricultural losses. In this study, a highly sensitive electrochemical immunosensor was developed based on both direct and sandwich immunoassays for the detection of TBRFV. The assay employs TBRFV-CP-IgG and TBRFV-CP-IgGHRP antibodies, with the latter conjugated to horseradish peroxidase (HRP). The immunoassays were assembled on a nanoporous gold electrode, providing an enhanced electroactive surface for efficient antigen capture and signal amplification. Electrochemical characterization confirmed the successful immobilization of TBRFV-CP-IgG, its specific interaction with the recombinant coat protein of TBRFV (rp-CP-TBRFV), the subsequent binding of TBRFV-CP-IgGHRP as the detection antibody, and the formation of the complete sandwich complex. The assays achieved linear detection ranges of 10–10⁵ fg/mL. The direct assay yielded a limit of detection (LoD) of 1.14 fg/mL (65.14 aM), while the sandwich assay, enhanced by enzymatic amplification, achieved 1.06 fg/mL (60.57 aM). The direct assay’s simplicity suits rapid diagnostics, whereas the sandwich assay’s superior sensitivity is ideal for low-concentration samples. Electrochemical characterization confirmed specific antigen capture and signal amplification. The biosensor demonstrated high specificity, distinguishing TBRFV from other viruses, and detected TBRFV in leaf and seed extracts, offering a promising platform for agricultural biosecurity. |
| format | Article |
| id | doaj-art-0e922a51df7b4635bcb52fb75dd0bc36 |
| institution | Kabale University |
| issn | 1746-4811 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | BMC |
| record_format | Article |
| series | Plant Methods |
| spelling | doaj-art-0e922a51df7b4635bcb52fb75dd0bc362025-08-20T03:45:32ZengBMCPlant Methods1746-48112025-07-0121111210.1186/s13007-025-01407-3Attomolar electrochemical direct and sandwich immunoassays for the ultrasensitive detection of tomato brown rugose fruit virusNegin Rezaei0Ahmad Moshaii1Mohammad Reza Safarnejad2Reza H. Sajedi3Mahsa Rahmanipour4Masoud Shams-Bakhsh5Plant Pathology Department, Faculty of Agriculture, Tarbiat Modares University (TMU)Department of Physics, Tarbiat Modares UniversityDepartment of Plant Viruses, Iranian Research Institute of Plant Protection, Agricultural research, education and extension organization (AREEO)Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares UniversityDepartment of Physics, Tarbiat Modares UniversityPlant Pathology Department, Faculty of Agriculture, Tarbiat Modares University (TMU)Abstract Tomato brown rugose fruit virus (TBRFV; Tobamovirus fructirugosum) is a highly virulent tobamovirus that has emerged as a major global threat to tomato and pepper crops over the past decade. Early and ultra-sensitive detection of TBRFV is critical for effective disease management and the mitigation of agricultural losses. In this study, a highly sensitive electrochemical immunosensor was developed based on both direct and sandwich immunoassays for the detection of TBRFV. The assay employs TBRFV-CP-IgG and TBRFV-CP-IgGHRP antibodies, with the latter conjugated to horseradish peroxidase (HRP). The immunoassays were assembled on a nanoporous gold electrode, providing an enhanced electroactive surface for efficient antigen capture and signal amplification. Electrochemical characterization confirmed the successful immobilization of TBRFV-CP-IgG, its specific interaction with the recombinant coat protein of TBRFV (rp-CP-TBRFV), the subsequent binding of TBRFV-CP-IgGHRP as the detection antibody, and the formation of the complete sandwich complex. The assays achieved linear detection ranges of 10–10⁵ fg/mL. The direct assay yielded a limit of detection (LoD) of 1.14 fg/mL (65.14 aM), while the sandwich assay, enhanced by enzymatic amplification, achieved 1.06 fg/mL (60.57 aM). The direct assay’s simplicity suits rapid diagnostics, whereas the sandwich assay’s superior sensitivity is ideal for low-concentration samples. Electrochemical characterization confirmed specific antigen capture and signal amplification. The biosensor demonstrated high specificity, distinguishing TBRFV from other viruses, and detected TBRFV in leaf and seed extracts, offering a promising platform for agricultural biosecurity.https://doi.org/10.1186/s13007-025-01407-3TBRFVElectrochemical immunosensorHorseradish peroxidaseSandwich immunoassays |
| spellingShingle | Negin Rezaei Ahmad Moshaii Mohammad Reza Safarnejad Reza H. Sajedi Mahsa Rahmanipour Masoud Shams-Bakhsh Attomolar electrochemical direct and sandwich immunoassays for the ultrasensitive detection of tomato brown rugose fruit virus Plant Methods TBRFV Electrochemical immunosensor Horseradish peroxidase Sandwich immunoassays |
| title | Attomolar electrochemical direct and sandwich immunoassays for the ultrasensitive detection of tomato brown rugose fruit virus |
| title_full | Attomolar electrochemical direct and sandwich immunoassays for the ultrasensitive detection of tomato brown rugose fruit virus |
| title_fullStr | Attomolar electrochemical direct and sandwich immunoassays for the ultrasensitive detection of tomato brown rugose fruit virus |
| title_full_unstemmed | Attomolar electrochemical direct and sandwich immunoassays for the ultrasensitive detection of tomato brown rugose fruit virus |
| title_short | Attomolar electrochemical direct and sandwich immunoassays for the ultrasensitive detection of tomato brown rugose fruit virus |
| title_sort | attomolar electrochemical direct and sandwich immunoassays for the ultrasensitive detection of tomato brown rugose fruit virus |
| topic | TBRFV Electrochemical immunosensor Horseradish peroxidase Sandwich immunoassays |
| url | https://doi.org/10.1186/s13007-025-01407-3 |
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