Attomolar Electrochemical Direct and Sandwich Immunoassays for the Ultrasensitive Detection of Tomato Brown Rugose Fruit Virus

Attomolar Electrochemical Direct and Sandwich Immunoassays for the Ultrasensitive Detection of Tomato Brown Rugose Fruit Virus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Attomolar Electrochemical Direct and Sandwich Immunoassays for the Ultrasensitive Detection of Tomato Brown Rugose Fruit Virus Negin Rezaei, Ahmad Moshaii, Mohammad Reza Safarnejad, Reza H. Sajedi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6247800/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Plant Methods → Version 1 posted 9 You are reading this latest preprint version 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-IgG HRP 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-IgG HRP as the detection antibody, and the formation of the complete sandwich complex. Upon exposure to the TMB/H₂O₂ substrate, HRP-catalyzed redox reactions generated a measurable electrochemical signal, enabling precise quantification of TBRFV. The developed biosensor exhibited a wide linear detection range from 0 to 10 5 fg/mL, with an ultra-low detection limit of 1.14 fg/mL, corresponding to 65.14 aM. Furthermore, the sensor demonstrated high specificity for TBRFV, effectively distinguishing it from potential interfering agents. The proposed electrochemical immunosensing strategy provides a highly promising platform for the early and accurate detection of TBRFV, offering significant potential for agricultural biosecurity and the prevention of viral outbreaks. TBRFV Electrochemical immunosensor Horseradish peroxidase Sandwich immunoassays Figures Figure 1 Figure 2 Figure 3 1. Introduction The tomato brown rugose fruit virus (TBRFV; Tobamovirus fructirugosum) is an emerging tobamovirus with a positive-sense single-stranded RNA genome containing four open reading frames (ORFs). It belongs to the Virgaviridae family and the Tobamovirus genus. Over the past decade, TBRFV has rapidly spread worldwide, reaching pandemic levels with a high prevalence rate. It was first reported in Jordan in 2015 [ 1 ] and has since been detected across all continents [ 2 , 3 ]. The virus primarily infects Solanaceae species, particularly tomatoes and peppers, posing a severe threat to global crop production. Notably, resistance genes such as Tm (in tomatoes) and L (in peppers), which confer resistance against related viruses, have proven ineffective against TBRFV [ 4 , 5 ]. TBRFV is primarily transmitted through direct contact between infected and healthy plants, contaminated seeds, agricultural tools, equipment, and human-mediated transfer (via contaminated hands or clothing). This high transmissibility facilitates long-distance spread [ 6 ]. Additionally, some insect vectors may contribute to its dissemination. The hallmark symptoms of TBRFV infection include mosaic patterns, leaf narrowing, dark green blistering on leaves, and brown, wrinkled spots on fruits, significantly reducing the marketability of affected crops [ 1 , 7 ]. Given its high environmental stability and extensive transmission potential, strict hygiene protocols, equipment disinfection, and the use of virus-free seeds are crucial for containment. Studies indicate that TBRFV can reduce crop yields by 15–55%, even in tomato plants carrying the Tm-22 resistance gene [ 8 ]. Thus, the development of highly sensitive and precise diagnostic methods is essential for effective virus detection and management. Among various diagnostic approaches, electrochemical immunosensors have attracted significant attention due to their high sensitivity, low detection limits, and cost-effectiveness [ 9 , 10 ]. These biosensors function by immobilizing specific antibodies onto a sensitive transducer surface, where they selectively interact with the target antigen, generating a measurable electrochemical response. This allows for rapid, highly specific detection of viral antigens, even at ultra-low concentrations, in complex biological samples [ 11 , 12 ]. Studies have shown that integrating nanostructured materials into electrochemical biosensors—such as nanocomposites [ 13 ], graphene [ 14 ], silver nanoparticles [ 15 ], and gold nanostructures [ 16 ] enhances the effective electroactive surface area, facilitates charge transfer, and significantly improves sensor sensitivity. Among these, gold nanostructures are particularly advantageous due to their high electrical conductivity, biocompatibility, and resistance to oxidation [ 17 ]. Electrochemical biosensors can be designed in a sandwich assay format, incorporating a secondary antibody conjugated to an electroactive enzyme, such as horseradish peroxidase (HRP). Upon exposure to 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H₂O₂, HRP catalyzes redox reactions, leading to the electrochemical detection of the target analyte [ 18 ]. Previous studies have demonstrated that enzyme-labeled antibodies in electrochemical biosensors enhance signal amplification, increase sensitivity, and improve detection performance [ 19 , 20 , 21 , 22 ]. This sandwich electrochemical immunosensor approach has been successfully applied in detecting various pathogens and biomarkers, including Salmonella serotypes [ 23 ], exosomes [ 24 ], Enterovirus 71 [ 18 ], carcinoembryonic antigen (CEA) [ 9 , 25 ], and the CA15-3 tumor marker [ 26 ]. Additionally, similar biosensing platforms have been utilized for detecting plant viruses, including cucumber mosaic virus (CMV) [ 27 ], rice tungro disease (RTD) [ 28 ], and citrus tristeza virus (CTV) [ 29 ]. The present study aims to develop a highly sensitive and specific electrochemical immunosensing platform for the ultrasensitive detection of TBRFV using both direct and sandwich-format immunoassays. The biosensor employs electrodes modified with nanoporous gold nanostructures [ 30 ]. The immunosensing system utilizes TBRFV-CP-IgG and TBRFV-CP-IgG HRP antibodies, along with the recombinant TBRFV coat protein antigen (rp-CP-TBRFV). Electrochemical characterization was performed using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV) in the presence of the TMB/H₂O₂ substrate. The developed biosensor exhibited a broad linear detection range, with an ultra-low detection limit of 1.14 fg/mL (equivalent to 65.14 attomolar), demonstrating high specificity and excellent reproducibility. Furthermore, the sensor successfully detected TBRFV in leaf tissue extracts (up to 1:256 dilution) and seed extracts (up to 1:2 dilution). This study introduces a highly promising electrochemical biosensing strategy for TBRFV detection, providing a powerful tool for plant biosecurity and effective virus management, ultimately mitigating the widespread damage caused by this emerging pathogen. 2. Materials and Methods Reagents and Materials High-purity gold (99.99%) and silver (99.99%) metals, fluorine-doped tin oxide (FTO) substrates (15 Ω/sq, 0.8 × 1.25 cm², 2 mm thickness), and phosphate-buffered saline (PBS, pH 7.4) were used as fundamental materials. Chemicals including mercaptoacetic acid (MAA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were procured from Sigma-Aldrich. Additional reagents included gelatin, ethanol (99.9%), pure acetone (99.98%, Merck), recombinant TBRFV coat protein (rp-CP-TBRFV) expressed in Escherichia coli , NaH 2 PO 4 (50 mM), NaCl (300 mM), imidazole (10 mM), lysozyme, TBRFV-CP-IgG and TBRFV-CP-IgG HRP antibodies, and TMB/H 2 O 2 substrate. Electrochemical measurements were conducted using potassium chloride (KCl), potassium ferricyanide (K 3 [Fe(CN) 6 ]), potassium ferrocyanide (K 4 [Fe(CN) 6 ]), and deionized water (DI water). All reagents were used without further purification. Electrochemical Instrumentation : Electrochemical measurements, including CV, EIS, and DPV, were carried out using the Origalysis potentiostat system (ElectroChem SAS, France). The system employed a three-electrode configuration: an Ag/AgCl reference electrode, a gold plate counter electrode, and a nanoporous gold-modified working electrode. Expression and Purification of rp-CP-TBRFV : The recombinant TBRFV coat protein was expressed and purified as follows: The CP gene, fused with a His-tag, was cloned into the pET-28a (+) bacterial expression vector and transformed into E. coli BL21. Expression was induced in a 1 L culture at 28°C using 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 3 hours under continuous shaking at 180 rpm. The bacterial pellet was resuspended in lysis buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, pH 8.0) containing 1 mg/mL lysozyme, followed by eight cycles of sonication (30-second pulses at 182 W with 30-second intervals). Purification was conducted under native conditions using immobilized metal ion affinity chromatography (IMAC) per the manufacturer’s instructions (Qiagen, Netherlands). SDS-PAGE analysis confirmed the purity and expression of recombinant TBRFV-CP, revealing a distinct band at approximately 23 kDa. Fabrication of the Sensing Electrode Electrode preparation and surface modification followed the protocol described by Yarjou et al. [ 30 ]. Initially, FTO electrodes were thoroughly cleaned with acetone, ethanol (99.9%), and DI water, followed by ultrasonication for 15 minutes and subsequent drying in an oven. A thin layer of 5 nm silver and 5 nm gold was sequentially deposited onto the FTO electrodes using physical vapor deposition (PVD) under a vacuum of 1 × 10⁻⁶ Torr. The deposition rate was maintained at 0.1 nm/s, with layer thickness monitored via a quartz crystal microbalance. Nanoporous Ag-Au alloy nanostructures were obtained by thermally annealing the electrodes at 550°C for 2 hours. The dealloying process was conducted by immersing the annealed electrodes in 65% nitric acid at room temperature for 15 minutes, followed by extensive rinsing with DI water. Biofunctionalization of the Sensor : For biofunctionalization, the gold surface was initially treated with 50 µL of 14 mmol/L MAA solution for 2 hours at room temperature, followed by ethanol (65%) washing. Carboxyl group activation was achieved using 50 µL of 50 mM EDC/NHS (1:1) in PBS (pH 4.5) for 1 hour at room temperature. Next, 50 µL of 10 µg/mL TBRFV-CP-IgG was immobilized onto the activated electrode and incubated overnight at 4°C to ensure stable antibody attachment. To establish the sandwich assay, blocking was performed using 50 µL of 20 mg/mL gelatin in PBS for 45 minutes. Various concentrations of rp-CP-TBRFV were incubated separately on individual working electrodes for 45 minutes at 4°C. Subsequently, a 1:5000 dilution of TBRFV-CP-IgGHRP with 50 µL of 0.42 µg/mL TBRFV-CP-IgGHRP was added and incubated for 1 hour at 4°C [ 19 ]. Between each step, electrodes were rinsed thoroughly with PBS (pH 7.4). To prevent photochemical degradation of MAA thiol groups, all functionalization steps were conducted in a dark environment. Electrochemical Measurements Electrochemical characterization was performed in a 20 mL electrolyte solution containing 2.5 mM [Fe(CN) 6 ] ⁻³/⁻⁴ and 0.1 M KCl, using an Ag/AgCl reference electrode. Each measurement was conducted in triplicate. CV was recorded from − 300 mV to 450 mV at a scan rate of 10 mV/s. EIS was measured across a frequency range of 0.1 Hz to 10⁵ Hz (Zre vs. Zim at 160 mV vs. Ag/AgCl reference). DPV measurements were recorded within a range of 10 µA to 100 µA. All electrochemical measurements were carried out at room temperature. The final electrochemical detection was performed following the formation of the sandwich complex in the presence of TMB (15 µM) and H 2 O 2 (20 µM) in 0.1 M KCl [ 31 ]. Performance and Selectivity of the Immunosensor : To evaluate the immunosensor’s performance, a calibration curve was generated, and the limit of detection (LoD) was determined using serial dilutions of rp-CP-TBRFV in PBS (pH 7.4), ranging from 0 to 10⁵ fg/mL. Furthermore, the sensor's selectivity was assessed in the presence of potential interfering agents from real samples. This included serial dilutions of infected leaf tissue extract (1:64 to 1:512), infected seed extract (1:2 to 1:4), and leaf extracts containing Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), and Tomato yellow leaf curl virus (TYLCV). Extracts from virus-free leaf and seed samples served as negative controls. 3. Results and Discussion The detection of TBRFV with the developed immunosensor system can be carried out using two sensing approaches. The first is direct measurement of the viral coat protein and the second is the sandwich-assay of the protein based on an HRP-labeled antibody as the enzyme and TMB/H₂O₂ as the substrate. For both sensing approaches, the modified electrode surface was functionalized using MAA, EDC, and NHS to activate covalent bonding [ 30 , 32 ] and ensure stable immobilization of the TBRFV-CP-IgG antibody. The immunosensor characteristics were assessed using CV, EIS, and DPV. Prior to the immobilization of TBRFV-CP-IgG, the electrode surface was characterized in its bare state, without sensing elements. The immobilization process involved TBRFV-CP-IgG attachment, blocking with gelatin to fill any unoccupied surface sites [ 33 ], the addition of rp-CP-TBRFV as the antigen, and TBRFV-CP-IgG HRP as the enzyme-labeled antibody. Finally, to evaluate the immunocomplex interaction, the enzymatic substrate TMB/H₂O₂ was introduced. Electrochemical measurements were performed after each step of immunocomplex formation and substrate addition and the results have been shown in Fig. 1 . Figure 1 a presents CV measurements conducted at a scan rate of 50 mV/s, illustrating the electrochemical behavior of the biosensor during its fabrication and functionalization stages. The analysis focuses on six key stages: the bare electrode, antibody immobilization, gelatin blocking, viral antigen binding, secondary antibody attachment, and enzymatic substrate reaction. The CV of the bare electrode (Porous Ag-Au Alloy) shows a high anodic peak current of 705 µA, indicating robust electron transfer at the pristine nanoporous silver-gold alloy surface. This high conductivity reflects the electrode’s clean, unmodified state, where redox-active species in the electrolyte (e.g., [Fe(CN) 6 ] 3−/4− ) can freely access the surface, facilitating efficient oxidation and reduction reactions. Upon immobilizing TBRFV-specific antibodies at a concentration of 10 µg/mL, the anodic peak current decreases sharply to 560 µA. This reduction arises because the antibody layer introduces steric hindrance and insulating properties, obstructing electron transfer between the electrode and the electrolyte. The drop in current confirms successful antibody attachment, as the biomolecules block the electrode’s active sites, reducing its electrochemical activity. After blocking unoccupied surface sites with gelatin, the current decreases further (to 541 µA). Gelatin acts as a biocompatible barrier, preventing nonspecific binding of unwanted molecules. This step is critical for enhancing selectivity, as it ensures that subsequent interactions are specific to the target antigen. However, the additional insulating layer further restricts electron transfer, amplifying the resistance observed in earlier stages. The introduction of the viral antigen (rp-CP-TBRFV) at 1 pg/mL causes the current to decline further (to 520 µA). This reduction occurs because the antigen binds to the immobilized antibodies, forming the first layer of the immunocomplex. The biomolecular interaction increases surface resistance, further impeding electron transfer and signaling the successful capture of the target analyte. Adding the HRP-labeled secondary antibody (TBRFV-CP-IgG HRP ) at 0.42 µg/mL results in a further drop in current (to 513 µA). The secondary antibody binds to the antigen, creating a thicker immunocomplex layer. This amplifies steric hindrance and insulating effects, reducing electrochemical activity. This step is essential for signal amplification in the subsequent enzymatic reaction. Upon exposure to the TMB/H₂O₂ substrate, the anodic peak current increases dramatically to 627 µA, nearly restoring it to the initial bare electrode value (705 µA). This recovery occurs because HRP catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by hydrogen peroxide (H₂O₂), generating a soluble redox-active product (TMB⁺). This product shuttles electrons to the electrode, bypassing the insulating immunocomplex layers. The sharp increase in current validates the enzymatic amplification strategy, which is pivotal for achieving ultra-low detection limits (e.g., 1.06 fg/mL). The CV analysis on the aforementioned six key stages demonstrates how the biosensor dynamically modulates electron transfer through bio-recognition events and enzymatic amplification. Figure 1 b presents DPV measurements, which provide higher sensitivity to subtle changes in electrochemical activity compared to CV. The results track the biosensor’s response across its fabrication and functionalization stages, with the x-axis representing applied potential and the y-axis showing current density (µA/cm²). The initial peak current density for the bare electrode is 234 µA/cm², reflecting unimpeded electron transfer at the pristine electrode surface. This high conductivity aligns with the CV results, confirming the electrode’s optimal performance in its unmodified state. After antibody attachment, the current density decreases to 205 µA/cm². This reduction is attributed to the insulating properties of the antibody layer, which blocks direct electron transfer between the electrode and the redox probe ([Fe(CN) 6 ]³⁻/⁴⁻). The introduction of the viral antigen (1 pg/mL) causes a further decline to 150 µA/cm². This drop confirms the formation of the antigen-antibody immunocomplex, which increases surface resistance and restricts electron transfer. After immobilizing the HRP-labeled secondary antibody (TBRFV-CP-IgG HRP ), the current density decreases to 140 µA/cm². The thicker immunocomplex layer amplifies steric hindrance and insulation, further reducing electrochemical activity. Upon exposure to the substrate, enzymatic reaction with TMB/H₂O₂ occurs and the current density surges to 220 µA/cm², nearing the bare electrode’s value (234 µA/cm²). The DPV results corroborate the CV findings, validating the enzymatic amplification strategy for enhancing sensitivity. Figure 1 c displays the EIS data, which measures charge transfer resistance (R ct ) at the electrode-electrolyte interface. The Nyquist plots clearly reveal how each functionalization step alters surface resistance. The initial R ct of the bare electrode is 16 Ω, indicative of a clean, conductive surface with minimal resistance to electron transfer. The R ct increases to 22 Ω after the first antibody attachment. After the gelatin blocking and antigen binding, further resistance increases are observed as gelatin and antigen layers add steric hindrance. The R ct for the secondary antibody immobilization is about 141 Ω, reflecting the cumulative insulating effect of the immunocomplex layers. Post-substrate addition of TMB/H 2 O 2 , results in R ct plummets to 20 Ω, nearly matching the bare electrode’s resistance. This dramatic reduction occurs because TMB⁺, generated by HRP-catalyzed oxidation, acts as a redox mediator, restoring efficient electron transfer. The EIS results complement the CV and DPV data, demonstrating how the biosensor’s resistance dynamically responds to bio-recognition events and enzymatic reactions. This triad of electrochemical techniques collectively validates the sensor’s design, enabling femtogram-level sensitivity and specificity for TBRFV detection. It should be mentioned that the trend of our findings in Fig. 1 align well with previous reports in the literature [ 18 , 19 , 31 ]. The CV measurements in Fig. 1 a, indicated that the anodic peak current of the bare electrode surface was 705 µA, which decreased to 560 µA upon TBRFV-CP-IgG immobilization. This current further declined progressively with each step of sandwich immunocomplex formation. However, upon the addition of the enzymatic substrate, HRP catalyzed the reaction, reversing the declining trend. The peak current increased from 510 µA at the TBRFV-CP-IgG HRP stage to 627 µA, which was higher than the peak current observed after TBRFV-CP-IgG immobilization and nearly restored to the initial peak current value (705 µA) of the bare electrode before antibody immobilization (Fig. 1 a). Similarly, DPV results in Fig. 1 b demonstrated a decrease in peak current density from 234 µA/cm² in the bare electrode state to 205 µA/cm² after TBRFV-CP-IgG immobilization, further declining to 150 µA/cm² under addition of rp-CP-TBRFV as the antigen, and to 140 µA/cm² upon TBRFV-CP-IgG HRP binding. Consistent with the CV results, exposure to TMB/H₂O₂ led to an increase in current, reaching 220 µA/cm², which was again higher than the TBRFV-CP-IgG immobilization stage and close to the bare electrode current density. EIS measurements were also conducted to assess the surface resistance of the FTO electrode, as shown in Fig. 1 c. The resistance increased from 22 Ω after TBRFV-CP-IgG immobilization to 141 Ω upon TBRFV-CP-IgG HRP attachment. This incremental resistance trend was observed at each stage of sandwich immunocomplex formation. However, after the addition of TMB/H₂O₂, the resistance increase was halted, and the value reverted to 20 Ω. The observed decrease in current (CV and DPV) and increase in resistance (EIS) upon TBRFV-CP-IgG attachment, relative to the bare electrode, confirmed the successful immobilization of TBRFV-CP-IgG on the electrode surface. The subsequent decline in current and rise in resistance from TBRFV-CP-IgG to TBRFV-CP-IgG HRP stages indicated successful sandwich immunocomplex formation. However, in all measurements, the introduction of TMB/H₂O₂ reversed these trends, restoring the electrode close to its initial state before antibody immobilization. This phenomenon is attributed to an electrochemical reaction catalyzed by HRP in the presence of TMB/H₂O₂. TMB functions as an electroactive mediator that can undergo direct reduction on the electrode surface. HRP first catalyzes the reduction of H₂O₂, leading to the oxidation of HRP (HRP Ox ). This oxidized HRP is subsequently reduced by the chemically oxidized TMB (TMB Red ), converting HRP Ox ) to its reduced form (HRP Red ). Meanwhile, TMB Red is regenerated to its oxidized form (TMB Ox ) through interaction with H₂O₂, releasing two water molecules. Finally, TMB Ox undergoes electrochemical reduction on the electrode surface, enhancing the reduction current. It should be mentioned that our findings in Fig. 1 align well with previous reports in the literature [ 18 , 19 , 31 ]. Figure 2 provides a detailed electrochemical characterization of the developed immunosensor for detecting TBRFV using three distinct biosensing strategies: direct immunoassay, sandwich immunoassay, and enzymatic immunoassay. The figure is divided into six panels (a–f), each presenting EIS responses and corresponding calibration curves across a concentration range of 0 to 10 5 fg/mL of the coat protein rp-CP-TBRFV. The EIS response for direct immunoassay are shown in Figs. 2a and 2b assessing the sensor’s performance in detecting rp-CP-TBRFV through direct antigen-antibody interactions. The ΔR ct increases progressively with antigen concentration. At 0 fg/mL (negative control), ΔRct ≈ 22 Ω (baseline). At 10⁵ fg/mL, ΔR ct rises to ~ 141Ω. A linear relationship is observed between ΔR ct and the logarithm of rp-CP-TBRFV concentration. The regression coefficient (R²) is 0.99, indicating strong linearity and reliability. Calculating LoD using the formula LoD = S/3.3σ, where σ is the standard deviation of blank signals (n = 3 replicates), and S is calibration curve slope, results in the LoD of the direct immunoassay as low as 1.14 fg/mL (equivalent to 65.14 aM). To enhance sensitivity, a sandwich immunoassay format is employed, as shown in Fig. 2c. The dual-antibody approach amplifies the signal, as evidenced by a steeper increase in ΔRct compared to the direct assay. At 10⁵ fg/mL, ΔRct reaches ~ 210 Ω, reflecting the cumulative insulating effect of the antigen and secondary antibody layers. The calibration curve in Fig. 2d demonstrates a linear relationship (R² = 0.97) between ΔRct and antigen concentration, with a calculated LoD of 1.06 fg/mL. This result demonstrates superior sensitivity due to the sandwich format, which enhances antigen-antibody interactions and the signal output. The enzymatic amplification strategy is showcased in Fig. 2e. After forming the sandwich complex, the TMB/H₂O₂ substrate is introduced. Consequently, ΔRct decreases dramatically, reverting from ~ 210 Ω (post-secondary antibody) to ~ 20 Ω (near baseline) at 10⁵ fg/mL. This reversal confirms the efficacy of enzymatic amplification in overcoming steric hindrance. Figure 2f quantifies this response, showing a linear decrease in ΔRct with increasing antigen concentration (R² = 0.97). Fig. 3 demonstrates the practical applicability, sensitivity, and selectivity of the developed electrochemical immunosensor for detecting TBRFV in real agricultural samples. The figure comprises three panels (a, b, c), showcasing the sensor’s performance in infected leaf and seed extracts, calibration curves, and cross-reactivity tests against non-target viruses. Figure 3 a presents the electrochemical response curves for a dilution series of TBRFV-infected leaf extracts, ranging from 1:64 to 1:512 dilutions. The immunosensor employs the direct immunoassay format, where TBRFV-specific antibodies (TBRFV-CP-IgG) capture the viral antigen (rp-CP-TBRFV) in the samples. The results reveal a concentration-dependent decrease in current intensities, with detectable signals even at the highest dilution (1:512). This indicates the sensor’s ability to identify ultra-low viral concentrations in complex biological matrices. The progressive signal attenuation correlates with reduced antigen availability at higher dilutions, validating the assay’s dynamic range. Figure 3 b illustrates the corresponding calibration curve for the leaf extract dilution series. The plot exhibits a linear relationship between the electrochemical signal (ΔR ct ) and the logarithm of the dilution factor, with a regression coefficient (R²) of 0.99. This strong linearity confirms the sensor’s reliability and reproducibility in quantifying TBRFV across varying concentrations. Figure 3 c evaluates the immunosensor’s selectivity by testing its response to TBRFV-infected leaf and seed extracts versus negative controls and samples containing non-target viruses (Tobacco mosaic virus [TMV], Cucumber mosaic virus [CMV], Tomato yellow leaf curl virus [TYLCV]). The results show that in the leaf extracts, TBRFV is detectable at dilutions up to 1:256, confirming robust performance in complex plant matrices. While in the seed extracts, the detection is achieved at 1:2 dilutions, highlighting the sensor’s utility in screening infected seeds, a critical pathway for viral transmission. In addition, the sensor exhibits negligible cross-reactivity (< 10% of the TBRFV signal) with TMV, CMV, TYLCV, or virus-free samples. This specificity ensures reliable differentiation of TBRFV from phylogenetically related viruses, such as TMV (a tobamovirus like TBRFV). Table 1 provides a comprehensive comparison of the analytical capabilities of various electrochemical sandwich immunoassays documented in the literature, highlighting the performance metrics of the developed TBRFV immunosensor in the context of existing biosensing platforms. The table illustrates the linear detection ranges, detection limits (LoD), and references for several biosensors targeting different pathogens and biomarkers, including Enterovirus 71, carcinoembryonic antigen (CEA), and plant viruses such as cucumber mosaic virus (CMV) and citrus tristeza virus (CTV). This comparison underscores the versatility of electrochemical biosensors in diverse fields, from medical diagnostics to agricultural biosecurity, while highlighting the superior sensitivity and performance of the developed TBRFV immunosensor. Notably, the TBRFV immunosensor developed in this study demonstrates a remarkable linear detection range from 10 to 105 fg/mL, with an ultra-low LoD of 1.14 fg/mL (equivalent to 65.14 aM) for the direct assay and 1.06 fg/mL (equivalent to 60.57 aM) for the sandwich assay. These values surpass the sensitivity of many previously reported biosensors, such as those for CEA (LoD of 0.2 pg/mL) and CMV (LoD of 0.1 mg/mL), underscoring the exceptional sensitivity of the proposed platform. The integration of nanoporous gold nanostructures and HRP-labeled antibodies in the sandwich assay format significantly enhances signal amplification, enabling femtogram-level detection of TBRFV. This comparison not only validates the superior performance of the developed immunosensor but also positions it as a highly competitive tool for ultrasensitive detection of plant viruses, with potential applications in agricultural diagnostics and biosecurity. Table 1 Comparison of analytical performance metrics for various electrochemical sandwich immunoassays reported in the literature, including linear detection ranges, LoD, and detection methods. The table encompasses biosensors targeting plant viruses (e.g., cucumber mosaic virus and citrus tristeza virus) as well as human and animal pathogens (e.g., Enterovirus 71 and carcinoembryonic antigen), highlighting the superior sensitivity and performance of the developed TBRFV immunosensor. Biosensor substrate Detection method Linear range LoD Refs ITO/ AuNPs/ MUA/ EDC-NHS/ mAb/ EV71/ mAb-HRP CA* 0.1– 600 ng/mL 0.01 ng/mL [ 18 ] Anti-CEA/Ce-MoF@HA/Ag-HRP/GCE CA EIS 0.001– 80 ng/mL 0.2 pg/mL [ 9 ] CS-NG/GCE /Au@Ag/HRP-anti-CEA DPV 0.0001– 100 ng/mL 0.05 pg/mL [ 25 ] SPCE/ Anti- CMV/ CMV/ Anti-CMV-HRP CA 0.1–1.3 mg/mL 0.1mg/mL [ 27 ] CP- CTV/ Ab 2 / MB/ HRP DPV 1.95– 10.0 × 10 3 fg/mL 0.3 fg/mL [ 29 ] MNP–HBsAb/HBsAg/HBsAb–HRP CV 0.001– 0.015 ng/ mL 0.9 pg/mL [ 36 ] Ab1/CEA/(DNA/(ZMPs-HRP-Ab2)n CV 0.008–200 ng/mL 5 pg/mL [ 37 ] AuC-HRP-anti-AFP/AFP-anti-AFP-O-MB-AuNPs/GCE DPV 0.005–20 ng/mL 1.5 pg/mL [ 38 ] Au nano-porous/MAA/EDC-NHS/TBRFV-CP-IgG/Gelatin/rp-CP-TBRFV/ TBRFV-CP-IgG HRP EIS CV DPV 10–10 5 fg/ mL Direct assay: 1.14 fg/mL Sandwich assay: 1.06 fg/mL This work *: Chronoamperometry 4. Conclusions In this study, we have successfully developed an ultrasensitive electrochemical immunosensor for the detection of TBRFV, leveraging both direct and sandwich immunoassay formats. The biosensor employs nanoporous gold-modified electrodes and HRP-labeled antibodies to achieve exceptional sensitivity and specificity. The electrochemical characterization, conducted through CV, EIS, and DPV, confirmed the successful immobilization of TBRFV-specific antibodies, the formation of antigen-antibody complexes, and the enzymatic amplification of the electrochemical signal. The developed immunosensor demonstrated an ultra-low detection limit of 1.14 fg/mL (equivalent to 65.14 attomolar), which surpasses the sensitivity of conventional detection methods such as ELISA and fluorescent quantum dot-based assays. This remarkable sensitivity is attributed to the efficient signal amplification provided by the HRP-catalyzed redox reaction in the presence of the TMB/H₂O₂ substrate. The biosensor also exhibited excellent specificity, effectively distinguishing TBRFV from other closely related plant viruses, including TMV, CMV, and TYLCV. This high specificity ensures reliable detection in complex agricultural matrices, reducing the risk of false positives. The practical applicability of the immunosensor was validated through its ability to detect TBRFV in infected leaf and seed extracts, even at extreme dilution factors (1:256 for leaf extracts and 1:2 for seed extracts). This capability is crucial for early detection and containment of TBRFV outbreaks, particularly in agricultural settings where the virus can spread rapidly through contaminated seeds, tools, and human contact. The sensor's robustness and reproducibility further underscore its potential as a reliable diagnostic tool for plant biosecurity. The integration of nanoporous gold nanostructures into the electrode design played a pivotal role in enhancing the sensor's performance. The high surface area and excellent electrical conductivity of the nanoporous gold facilitated efficient antigen capture and signal amplification, while the biocompatibility and resistance to oxidation ensured the stability of the immunosensor over time. The use of HRP-labeled antibodies in the sandwich assay format further enhanced the sensitivity, allowing for the detection of trace amounts of TBRFV in complex samples. This study not only presents a highly sensitive and specific method for TBRFV detection but also highlights the broader potential of electrochemical immunosensors in agricultural diagnostics. The developed platform offers a rapid, cost-effective, and field-deployable solution for the early detection of plant pathogens, which is critical for preventing widespread agricultural losses. Future work will focus on integrating this sensing platform into portable electrochemical devices, enabling real-time monitoring and on-site diagnostics in agricultural settings. This advancement could significantly enhance the ability to manage and mitigate the impact of emerging plant viruses, safeguarding global food security. In conclusion, the proposed electrochemical immunosensor represents a significant step forward in the field of plant virus detection. Its ultra-low detection limit, high specificity, and practical applicability make it a powerful tool for the early and accurate detection of TBRFV, offering a promising solution for the prevention and control of viral outbreaks in agriculture. The success of this approach also opens new avenues for the development of similar biosensors for other plant. Declarations Authors’ contribution N.R. carried out all experiments and wrote the original manuscript. A.M., M.S.B., M.R.S., and R.H.S. designed the study and provided essential materials. M.R. advises the project. A.M., M.S.B., M.R.S., and R.H.S. edited the manuscript. All authors read and approved the final manuscript. Funding declaration This work received institutional support from Tarbiat Modares University as part of its doctoral dissertation funding program. No specific grants or financial awards were provided by external public, commercial, or non-profit funding agencies for this research. Availability of data and materials Not applicable. Ethics and Consent to Participate declarations Not applicable. Consent to Publish declaration Not applicable Competing interests The authors declare no competing interests. References Salem, N., Mansour, A., Ciuffo, M., Falk, B. and Turina, M. A new tobamovirus infecting tomato crops in Jordan. Arch. Virol. 2016;161: 503-506. Mehle, N., Bačnik, K., Bajde, I., Brodarič, J., Fox, A., Gutiérrez-Aguirre, I., Kitek, M., Kutnjak, D., Loh, Y.L. and Maksimović Carvalho Ferreira, O. Tomato brown rugose fruit virus in aqueous environments–survival and significance of water-mediated transmission. Front. Plant Sci. 2023; 14: 1187920. 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A dual amplification electrochemical immunosensor based on HRP-Au@ Ag NPs for carcinoembryonic antigen detection. Anal. Biochem. 2019; 574: 23-30. Martins, T. S., Bott-Neto, J. L., Oliveira Jr, O. N., & Machado, S. A. A sandwich-type electrochemical immunosensor based on Au-rGO composite for CA15-3 tumor marker detection. Mikrochim. Acta. 2022; 189(1): 38. Rafidah, A., Faridah, S., Shahrul, A., Mazidah, M. and Zamri, I. Chronoamperometry measurement for rapid cucumber mosaic virus detection in plants. Procedia Chem. 2016; 20: 25-28. Uda, M., Hasfalina, C., Samsuzanaa, A., Faridah, S., Zamri, I., Noraini, B.S., Sabrina, W.N., Hashim, U. and Gopinath, S.C. Determination of fixed-potential selection using chronoamperometry analysis by screen-printed carbon electrode for rice tungro bacilliform virus (RTBV) detection. Paper presented at the AIP Conference Proceedings. 2017. Freitas, T.A., Proenca, C.A., Baldo, T.A., Materon, E.M., Wong, A., Magnani, R.F. and Faria, R.C. Ultrasensitive immunoassay for detection of Citrus tristeza virus in citrus sample using disposable microfluidic electrochemical device. Talanta. 2019; 205: 120110. Yarjoo, S., Siampour, H., Khalilipour, M., Sajedi, R.H., Bagheri, H. and Moshaii, A. Gold nanostructure-enhanced immunosensing: ultra-sensitive detection of VEGF tumor marker for early disease diagnosis. Sci. Rep. 2024; 14(1): 10450. Ahirwal, G.K. and Mitra, C.K. Gold nanoparticles based sandwich electrochemical immunosensor. Biosens. Bioelectron. 2010; 25(9): 2016-2020. Stanković, V., Đurđić, S., Ognjanović, M., Antić, B., Kalcher, K., Mutić, J. and Stanković, D.M. Anti-human albumin monoclonal antibody immobilized on EDC-NHS functionalized carboxylic graphene/AuNPs composite as promising electrochemical HSA immunosensor. J. Electroanal. Chem. 2020; 860: 113928. Miyake, S., Irikura, D. and Yamasaki, T. Detection of Mast Cells Expressing c-Kit Using Antibody Covalently Bound to Gelatin Elongated from Surface of Immunosensor Based on Surface Plasmon Resonance. Anal. Sci. 2019; 35(7): 811-813. Hayashi, Y., Matsuda, R., Ito, K., Nishimura, W., Imai, K. and Maeda, M. Detection limit estimated from slope of calibration curve: an application to competitive ELISA. Anal. Sci. 2005; 21(2): 167-169. Liu, J., Liu, B., Liu, J., He, X.-D., Yuan, J., Ghassemlooy, Z., Torun, H., Fu, Y.-Q., Dai, X. and Ng, W.P. Integrated label-free erbium-doped fiber laser biosensing system for detection of single cell Staphylococcus aureus. Talanta. 2023; 257: 124385. Nourani S, Ghourchian H, Boutorabi SM. Magnetic nanoparticle-based immunosensor for electrochemical detection of hepatitis B surface antigen. Anal. Biochem. 2013; 441(1): 1-7. Gan, N., Jia, L., & Zheng, L. A sandwich electrochemical immunosensor using magnetic DNA nanoprobes for carcinoembryonic antigen. Int. J. Mol. Sci. 2011; 12(11): 7410-7423. Shen, C., Wang, L., Zhang, H., Liu, S., & Jiang, J. An electrochemical sandwich immunosensor based on signal amplification technique for the determination of alpha-fetoprotein. Front. Chem. 2020; 8: 589560. Additional Declarations No competing interests reported. Supplementary Files Graphicalabstract.tif Cite Share Download PDF Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Plant Methods → Version 1 posted Editorial decision: Revision requested 01 May, 2025 Reviews received at journal 27 Apr, 2025 Reviewers agreed at journal 27 Apr, 2025 Reviews received at journal 21 Apr, 2025 Reviewers agreed at journal 02 Apr, 2025 Reviewers invited by journal 31 Mar, 2025 Editor assigned by journal 24 Mar, 2025 Submission checks completed at journal 24 Mar, 2025 First submitted to journal 17 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6247800","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":444260459,"identity":"8b8dd732-82cb-4f26-b520-7280276235dc","order_by":0,"name":"Negin Rezaei","email":"","orcid":"","institution":"Tarbiat Modares University (TMU)","correspondingAuthor":false,"prefix":"","firstName":"Negin","middleName":"","lastName":"Rezaei","suffix":""},{"id":444260460,"identity":"854257c5-a0a8-4503-9a29-63a0ddf60012","order_by":1,"name":"Ahmad Moshaii","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIie3PsQrCMBCA4RPBqeCaoNQnEFKEqG/TTl0UBEE6SEkRzOKrOIjgrATa5R6gbk6dXMRFQcS4iKBE3RzyT8fBx3EANtt/VgFgxNUDe6zW35DWrwQgEM/EWFPKYncadMK5nCyOURRDVa5LKjIQjtj2Zoz0V5gOKaICgj5s0ETyXoU4d5L3GE2m+oscYCOMJCzohZFQ29Y5ucbQ+Ex8XtNXfE04TUQZ2EeCyGt1Rjz9y6grUuV4GAgzyWRB95e4wbPJcivGsetmSh1M5CUHoPQTsNlsNtubbkBMT7JI6HPUAAAAAElFTkSuQmCC","orcid":"","institution":"Tarbiat Modares University (TMU)","correspondingAuthor":true,"prefix":"","firstName":"Ahmad","middleName":"","lastName":"Moshaii","suffix":""},{"id":444260461,"identity":"932fe60b-0eee-47cf-bcab-066916ebdd3b","order_by":2,"name":"Mohammad Reza Safarnejad","email":"","orcid":"","institution":"Iranian Research Institute of Plant Protection, Agricultural research, education and extension organization (AREEO)","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Reza","lastName":"Safarnejad","suffix":""},{"id":444260462,"identity":"d56e81ff-01aa-477d-8520-2d0bbe3104cd","order_by":3,"name":"Reza H. Sajedi","email":"","orcid":"","institution":"Tarbiat Modares University (TMU)","correspondingAuthor":false,"prefix":"","firstName":"Reza","middleName":"H.","lastName":"Sajedi","suffix":""},{"id":444260463,"identity":"ce58795d-2a15-4232-9c17-dbc2a167f118","order_by":4,"name":"Mahsa Rahmanipour","email":"","orcid":"","institution":"Tarbiat Modares University (TMU)","correspondingAuthor":false,"prefix":"","firstName":"Mahsa","middleName":"","lastName":"Rahmanipour","suffix":""},{"id":444260464,"identity":"4cb4e9d4-17df-4ef5-89a3-818ccc8b7da4","order_by":5,"name":"Masoud Shams-Bakhsh","email":"","orcid":"","institution":"Tarbiat Modares University (TMU)","correspondingAuthor":false,"prefix":"","firstName":"Masoud","middleName":"","lastName":"Shams-Bakhsh","suffix":""}],"badges":[],"createdAt":"2025-03-17 22:08:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6247800/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6247800/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13007-025-01407-3","type":"published","date":"2025-07-01T15:57:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81029755,"identity":"ecaaf025-ec52-46db-aff6-31d08c3a8e18","added_by":"auto","created_at":"2025-04-21 11:12:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":388488,"visible":true,"origin":"","legend":"\u003cp\u003eElectrochemical response graphs, including CV (a) at a scan rate of 50 mV/s, DPV (b), and EIS (c), corresponding to different stages of the biosensor formation. These stages include the porous electrode, antibody immobilization (at a concentration of 10 µg/mL), blocking with gelatin, addition of the viral surface protein (rp-CP-TBRFV) with concentration of 1 pg/mL, HRP-labeled secondary antibody with concentration of 0.42 µg/mL, and finally, exposure to the enzymatic substrate (TMB/H₂O₂).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6247800/v1/0bdeb4adf75f0e47791853f8.png"},{"id":81029756,"identity":"34927db3-854f-4ab5-a3b0-df74e6bbfed4","added_by":"auto","created_at":"2025-04-21 11:12:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":726824,"visible":true,"origin":"","legend":"\u003cp\u003eThe electrochemical characterization for the detection of TBRFV using different biosensing strategies. (a) EIS response for direct immunosensing at various concentrations of rp-CP-TBRFV. (b) Corresponding calibration curve demonstrating a linear relationship between ΔR\u003csub\u003ect\u003c/sub\u003e and the logarithm of rp-CP-TBRFV concentration. (c) EIS response for the sandwich immunoassay incorporating the HRP-labeled secondary antibody TBRFV-CP-IgG\u003csup\u003eHRP\u003c/sup\u003e. (d) Calibration curve showing enhanced signal amplification in the sandwich assay. (e) EIS response after enzymatic reaction with TMB/H₂O₂, demonstrating a decrease in charge transfer resistance. (f) Calibration curve illustrating the recovery of electrochemical activity following enzymatic catalysis. The results confirm that the sandwich immunoassay improves sensitivity compared to direct immunosensing, and the enzymatic reaction further enhances detection performance, making the developed biosensor a highly effective tool for TBRFV detection.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6247800/v1/b560cbf12a0d33030fc695b1.png"},{"id":81029757,"identity":"6e445494-6af0-40c0-b6c9-c5e4b2b92654","added_by":"auto","created_at":"2025-04-21 11:12:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":186023,"visible":true,"origin":"","legend":"\u003cp\u003eReal-sample detection and selectivity of the electrochemical immunosensor for TBRFV. (a) Electrochemical response curves for TBRFV detection in serially diluted infected leaf extracts (1:64 to 1:512), demonstrating concentration-dependent signal attenuation via direct immunoassay. (b) Calibration curve for leaf extract dilutions, showing a linear relationship (R² = 0.99) between charge transfer resistance (ΔR\u003csub\u003ect\u003c/sub\u003e) and logarithmic dilution factor. (c) Selectivity assessment: The immunosensor exhibits strong specificity for TBRFV in infected leaf (1:256 dilution) and seed extracts (1:2 dilution), with negligible cross-reactivity (\u0026lt;10% background signal) against non-target viruses (TMV, CMV, TYLCV) or virus-free controls. Results validate the sensor’s femtogram-level sensitivity, field-deployable utility, and robustness in complex agricultural matrices.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6247800/v1/49c2ac848fe25801319ca045.png"},{"id":86179615,"identity":"5b6fc6a9-df56-4809-9a73-557b148bc697","added_by":"auto","created_at":"2025-07-07 16:17:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1912905,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6247800/v1/f29828e8-b978-4ec8-ad01-f43517bdaf49.pdf"},{"id":81029759,"identity":"2f4111e0-ac94-45ba-9ba6-b77c6bcdadfb","added_by":"auto","created_at":"2025-04-21 11:12:26","extension":"tif","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":389386,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-6247800/v1/69ff1b598c2851c73825d528.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Attomolar Electrochemical Direct and Sandwich Immunoassays for the Ultrasensitive Detection of Tomato Brown Rugose Fruit Virus","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe tomato brown rugose fruit virus (TBRFV; Tobamovirus fructirugosum) is an emerging tobamovirus with a positive-sense single-stranded RNA genome containing four open reading frames (ORFs). It belongs to the Virgaviridae family and the Tobamovirus genus. Over the past decade, TBRFV has rapidly spread worldwide, reaching pandemic levels with a high prevalence rate. It was first reported in Jordan in 2015 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] and has since been detected across all continents [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The virus primarily infects Solanaceae species, particularly tomatoes and peppers, posing a severe threat to global crop production. Notably, resistance genes such as Tm (in tomatoes) and L (in peppers), which confer resistance against related viruses, have proven ineffective against TBRFV [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTBRFV is primarily transmitted through direct contact between infected and healthy plants, contaminated seeds, agricultural tools, equipment, and human-mediated transfer (via contaminated hands or clothing). This high transmissibility facilitates long-distance spread [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Additionally, some insect vectors may contribute to its dissemination. The hallmark symptoms of TBRFV infection include mosaic patterns, leaf narrowing, dark green blistering on leaves, and brown, wrinkled spots on fruits, significantly reducing the marketability of affected crops [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Given its high environmental stability and extensive transmission potential, strict hygiene protocols, equipment disinfection, and the use of virus-free seeds are crucial for containment. Studies indicate that TBRFV can reduce crop yields by 15\u0026ndash;55%, even in tomato plants carrying the Tm-22 resistance gene [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Thus, the development of highly sensitive and precise diagnostic methods is essential for effective virus detection and management.\u003c/p\u003e \u003cp\u003eAmong various diagnostic approaches, electrochemical immunosensors have attracted significant attention due to their high sensitivity, low detection limits, and cost-effectiveness [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. These biosensors function by immobilizing specific antibodies onto a sensitive transducer surface, where they selectively interact with the target antigen, generating a measurable electrochemical response. This allows for rapid, highly specific detection of viral antigens, even at ultra-low concentrations, in complex biological samples [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Studies have shown that integrating nanostructured materials into electrochemical biosensors\u0026mdash;such as nanocomposites [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], graphene [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], silver nanoparticles [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and gold nanostructures [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] enhances the effective electroactive surface area, facilitates charge transfer, and significantly improves sensor sensitivity. Among these, gold nanostructures are particularly advantageous due to their high electrical conductivity, biocompatibility, and resistance to oxidation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eElectrochemical biosensors can be designed in a sandwich assay format, incorporating a secondary antibody conjugated to an electroactive enzyme, such as horseradish peroxidase (HRP). Upon exposure to 3,3\u0026prime;,5,5\u0026prime;-tetramethylbenzidine (TMB) in the presence of H₂O₂, HRP catalyzes redox reactions, leading to the electrochemical detection of the target analyte [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Previous studies have demonstrated that enzyme-labeled antibodies in electrochemical biosensors enhance signal amplification, increase sensitivity, and improve detection performance [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. This sandwich electrochemical immunosensor approach has been successfully applied in detecting various pathogens and biomarkers, including Salmonella serotypes [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], exosomes [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], Enterovirus 71 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], carcinoembryonic antigen (CEA) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and the CA15-3 tumor marker [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Additionally, similar biosensing platforms have been utilized for detecting plant viruses, including cucumber mosaic virus (CMV) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], rice tungro disease (RTD) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and citrus tristeza virus (CTV) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe present study aims to develop a highly sensitive and specific electrochemical immunosensing platform for the ultrasensitive detection of TBRFV using both direct and sandwich-format immunoassays. The biosensor employs electrodes modified with nanoporous gold nanostructures [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The immunosensing system utilizes TBRFV-CP-IgG and TBRFV-CP-IgG\u003csup\u003eHRP\u003c/sup\u003e antibodies, along with the recombinant TBRFV coat protein antigen (rp-CP-TBRFV). Electrochemical characterization was performed using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV) in the presence of the TMB/H₂O₂ substrate. The developed biosensor exhibited a broad linear detection range, with an ultra-low detection limit of 1.14 fg/mL (equivalent to 65.14 attomolar), demonstrating high specificity and excellent reproducibility. Furthermore, the sensor successfully detected TBRFV in leaf tissue extracts (up to 1:256 dilution) and seed extracts (up to 1:2 dilution). This study introduces a highly promising electrochemical biosensing strategy for TBRFV detection, providing a powerful tool for plant biosecurity and effective virus management, ultimately mitigating the widespread damage caused by this emerging pathogen.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e \u003cstrong\u003eReagents and Materials\u003c/strong\u003e \u003cp\u003eHigh-purity gold (99.99%) and silver (99.99%) metals, fluorine-doped tin oxide (FTO) substrates (15 Ω/sq, 0.8 \u0026times; 1.25 cm\u0026sup2;, 2 mm thickness), and phosphate-buffered saline (PBS, pH 7.4) were used as fundamental materials. Chemicals including mercaptoacetic acid (MAA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were procured from Sigma-Aldrich. Additional reagents included gelatin, ethanol (99.9%), pure acetone (99.98%, Merck), recombinant TBRFV coat protein (rp-CP-TBRFV) expressed in \u003cem\u003eEscherichia coli\u003c/em\u003e, NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (50 mM), NaCl (300 mM), imidazole (10 mM), lysozyme, TBRFV-CP-IgG and TBRFV-CP-IgG\u003csup\u003eHRP\u003c/sup\u003e antibodies, and TMB/H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e substrate. Electrochemical measurements were conducted using potassium chloride (KCl), potassium ferricyanide (K\u003csub\u003e3\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e]), potassium ferrocyanide (K\u003csub\u003e4\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e]), and deionized water (DI water). All reagents were used without further purification.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eElectrochemical Instrumentation\u003c/b\u003e: Electrochemical measurements, including CV, EIS, and DPV, were carried out using the Origalysis potentiostat system (ElectroChem SAS, France). The system employed a three-electrode configuration: an Ag/AgCl reference electrode, a gold plate counter electrode, and a nanoporous gold-modified working electrode.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression and Purification of rp-CP-TBRFV\u003c/b\u003e: The recombinant TBRFV coat protein was expressed and purified as follows: The CP gene, fused with a His-tag, was cloned into the pET-28a (+) bacterial expression vector and transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21. Expression was induced in a 1 L culture at 28\u0026deg;C using 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 3 hours under continuous shaking at 180 rpm. The bacterial pellet was resuspended in lysis buffer (50 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 300 mM NaCl, 10 mM imidazole, pH 8.0) containing 1 mg/mL lysozyme, followed by eight cycles of sonication (30-second pulses at 182 W with 30-second intervals). Purification was conducted under native conditions using immobilized metal ion affinity chromatography (IMAC) per the manufacturer\u0026rsquo;s instructions (Qiagen, Netherlands). SDS-PAGE analysis confirmed the purity and expression of recombinant TBRFV-CP, revealing a distinct band at approximately 23 kDa.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFabrication of the Sensing Electrode\u003c/strong\u003e \u003cp\u003eElectrode preparation and surface modification followed the protocol described by Yarjou et al. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Initially, FTO electrodes were thoroughly cleaned with acetone, ethanol (99.9%), and DI water, followed by ultrasonication for 15 minutes and subsequent drying in an oven. A thin layer of 5 nm silver and 5 nm gold was sequentially deposited onto the FTO electrodes using physical vapor deposition (PVD) under a vacuum of 1 \u0026times; 10⁻⁶ Torr. The deposition rate was maintained at 0.1 nm/s, with layer thickness monitored via a quartz crystal microbalance. Nanoporous Ag-Au alloy nanostructures were obtained by thermally annealing the electrodes at 550\u0026deg;C for 2 hours. The dealloying process was conducted by immersing the annealed electrodes in 65% nitric acid at room temperature for 15 minutes, followed by extensive rinsing with DI water.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eBiofunctionalization of the Sensor\u003c/b\u003e: For biofunctionalization, the gold surface was initially treated with 50 \u0026micro;L of 14 mmol/L MAA solution for 2 hours at room temperature, followed by ethanol (65%) washing. Carboxyl group activation was achieved using 50 \u0026micro;L of 50 mM EDC/NHS (1:1) in PBS (pH 4.5) for 1 hour at room temperature. Next, 50 \u0026micro;L of 10 \u0026micro;g/mL TBRFV-CP-IgG was immobilized onto the activated electrode and incubated overnight at 4\u0026deg;C to ensure stable antibody attachment. To establish the sandwich assay, blocking was performed using 50 \u0026micro;L of 20 mg/mL gelatin in PBS for 45 minutes. Various concentrations of rp-CP-TBRFV were incubated separately on individual working electrodes for 45 minutes at 4\u0026deg;C. Subsequently, a 1:5000 dilution of TBRFV-CP-IgGHRP with 50 \u0026micro;L of 0.42 \u0026micro;g/mL TBRFV-CP-IgGHRP was added and incubated for 1 hour at 4\u0026deg;C [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Between each step, electrodes were rinsed thoroughly with PBS (pH 7.4). To prevent photochemical degradation of MAA thiol groups, all functionalization steps were conducted in a dark environment.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eElectrochemical Measurements\u003c/strong\u003e \u003cp\u003eElectrochemical characterization was performed in a 20 mL electrolyte solution containing 2.5 mM [Fe(CN)\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e⁻\u0026sup3;/⁻⁴\u003c/sup\u003e and 0.1 M KCl, using an Ag/AgCl reference electrode. Each measurement was conducted in triplicate. CV was recorded from \u0026minus;\u0026thinsp;300 mV to 450 mV at a scan rate of 10 mV/s. EIS was measured across a frequency range of 0.1 Hz to 10⁵ Hz (Zre vs. Zim at 160 mV vs. Ag/AgCl reference). DPV measurements were recorded within a range of 10 \u0026micro;A to 100 \u0026micro;A. All electrochemical measurements were carried out at room temperature. The final electrochemical detection was performed following the formation of the sandwich complex in the presence of TMB (15 \u0026micro;M) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (20 \u0026micro;M) in 0.1 M KCl [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePerformance and Selectivity of the Immunosensor\u003c/b\u003e: To evaluate the immunosensor\u0026rsquo;s performance, a calibration curve was generated, and the limit of detection (LoD) was determined using serial dilutions of rp-CP-TBRFV in PBS (pH 7.4), ranging from 0 to 10⁵ fg/mL. Furthermore, the sensor's selectivity was assessed in the presence of potential interfering agents from real samples. This included serial dilutions of infected leaf tissue extract (1:64 to 1:512), infected seed extract (1:2 to 1:4), and leaf extracts containing \u003cem\u003eTobacco mosaic virus\u003c/em\u003e (TMV), \u003cem\u003eCucumber mosaic virus\u003c/em\u003e (CMV), and \u003cem\u003eTomato yellow leaf curl virus\u003c/em\u003e (TYLCV). Extracts from virus-free leaf and seed samples served as negative controls.\u003c/p\u003e"},{"header":"3. Results and Discussion","content":"\u003cp\u003eThe detection of TBRFV with the developed immunosensor system can be carried out using two sensing approaches. The first is direct measurement of the viral coat protein and the second is the sandwich-assay of the protein based on an HRP-labeled antibody as the enzyme and TMB/H₂O₂ as the substrate. For both sensing approaches, the modified electrode surface was functionalized using MAA, EDC, and NHS to activate covalent bonding [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e] and ensure stable immobilization of the TBRFV-CP-IgG antibody. The immunosensor characteristics were assessed using CV, EIS, and DPV.\u003c/p\u003e\n\u003cp\u003ePrior to the immobilization of TBRFV-CP-IgG, the electrode surface was characterized in its bare state, without sensing elements. The immobilization process involved TBRFV-CP-IgG attachment, blocking with gelatin to fill any unoccupied surface sites [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e], the addition of rp-CP-TBRFV as the antigen, and TBRFV-CP-IgG\u003csup\u003eHRP\u003c/sup\u003e as the enzyme-labeled antibody. Finally, to evaluate the immunocomplex interaction, the enzymatic substrate TMB/H₂O₂ was introduced. Electrochemical measurements were performed after each step of immunocomplex formation and substrate addition and the results have been shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea presents CV measurements conducted at a scan rate of 50 mV/s, illustrating the electrochemical behavior of the biosensor during its fabrication and functionalization stages. The analysis focuses on six key stages: the bare electrode, antibody immobilization, gelatin blocking, viral antigen binding, secondary antibody attachment, and enzymatic substrate reaction. The CV of the bare electrode (Porous Ag-Au Alloy) shows a high anodic peak current of 705 \u0026micro;A, indicating robust electron transfer at the pristine nanoporous silver-gold alloy surface. This high conductivity reflects the electrode\u0026rsquo;s clean, unmodified state, where redox-active species in the electrolyte (e.g., [Fe(CN)\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e3\u0026minus;/4\u0026minus;\u003c/sup\u003e) can freely access the surface, facilitating efficient oxidation and reduction reactions.\u003c/p\u003e\n\u003cp\u003eUpon immobilizing TBRFV-specific antibodies at a concentration of 10 \u0026micro;g/mL, the anodic peak current decreases sharply to 560 \u0026micro;A. This reduction arises because the antibody layer introduces steric hindrance and insulating properties, obstructing electron transfer between the electrode and the electrolyte. The drop in current confirms successful antibody attachment, as the biomolecules block the electrode\u0026rsquo;s active sites, reducing its electrochemical activity. After blocking unoccupied surface sites with gelatin, the current decreases further (to 541 \u0026micro;A). Gelatin acts as a biocompatible barrier, preventing nonspecific binding of unwanted molecules. This step is critical for enhancing selectivity, as it ensures that subsequent interactions are specific to the target antigen. However, the additional insulating layer further restricts electron transfer, amplifying the resistance observed in earlier stages.\u003c/p\u003e\n\u003cp\u003eThe introduction of the viral antigen (rp-CP-TBRFV) at 1 pg/mL causes the current to decline further (to 520 \u0026micro;A). This reduction occurs because the antigen binds to the immobilized antibodies, forming the first layer of the immunocomplex. The biomolecular interaction increases surface resistance, further impeding electron transfer and signaling the successful capture of the target analyte. Adding the HRP-labeled secondary antibody (TBRFV-CP-IgG\u003csup\u003eHRP\u003c/sup\u003e) at 0.42 \u0026micro;g/mL results in a further drop in current (to 513 \u0026micro;A). The secondary antibody binds to the antigen, creating a thicker immunocomplex layer. This amplifies steric hindrance and insulating effects, reducing electrochemical activity. This step is essential for signal amplification in the subsequent enzymatic reaction.\u003c/p\u003e\n\u003cp\u003eUpon exposure to the TMB/H₂O₂ substrate, the anodic peak current increases dramatically to 627 \u0026micro;A, nearly restoring it to the initial bare electrode value (705 \u0026micro;A). This recovery occurs because HRP catalyzes the oxidation of 3,3\u0026apos;,5,5\u0026apos;-tetramethylbenzidine (TMB) by hydrogen peroxide (H₂O₂), generating a soluble redox-active product (TMB⁺). This product shuttles electrons to the electrode, bypassing the insulating immunocomplex layers. The sharp increase in current validates the enzymatic amplification strategy, which is pivotal for achieving ultra-low detection limits (e.g., 1.06 fg/mL). The CV analysis on the aforementioned six key stages demonstrates how the biosensor dynamically modulates electron transfer through bio-recognition events and enzymatic amplification.\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb presents DPV measurements, which provide higher sensitivity to subtle changes in electrochemical activity compared to CV. The results track the biosensor\u0026rsquo;s response across its fabrication and functionalization stages, with the x-axis representing applied potential and the y-axis showing current density (\u0026micro;A/cm\u0026sup2;). The initial peak current density for the bare electrode is 234 \u0026micro;A/cm\u0026sup2;, reflecting unimpeded electron transfer at the pristine electrode surface. This high conductivity aligns with the CV results, confirming the electrode\u0026rsquo;s optimal performance in its unmodified state. After antibody attachment, the current density decreases to 205 \u0026micro;A/cm\u0026sup2;. This reduction is attributed to the insulating properties of the antibody layer, which blocks direct electron transfer between the electrode and the redox probe ([Fe(CN)\u003csub\u003e6\u003c/sub\u003e]\u0026sup3;⁻/⁴⁻). The introduction of the viral antigen (1 pg/mL) causes a further decline to 150 \u0026micro;A/cm\u0026sup2;. This drop confirms the formation of the antigen-antibody immunocomplex, which increases surface resistance and restricts electron transfer.\u003c/p\u003e\n\u003cp\u003eAfter immobilizing the HRP-labeled secondary antibody (TBRFV-CP-IgG\u003csup\u003eHRP\u003c/sup\u003e), the current density decreases to 140 \u0026micro;A/cm\u0026sup2;. The thicker immunocomplex layer amplifies steric hindrance and insulation, further reducing electrochemical activity. Upon exposure to the substrate, enzymatic reaction with TMB/H₂O₂ occurs and the current density surges to 220 \u0026micro;A/cm\u0026sup2;, nearing the bare electrode\u0026rsquo;s value (234 \u0026micro;A/cm\u0026sup2;). The DPV results corroborate the CV findings, validating the enzymatic amplification strategy for enhancing sensitivity.\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec displays the EIS data, which measures charge transfer resistance (R\u003csub\u003ect\u003c/sub\u003e) at the electrode-electrolyte interface. The Nyquist plots clearly reveal how each functionalization step alters surface resistance. The initial R\u003csub\u003ect\u003c/sub\u003e of the bare electrode is 16 Ω, indicative of a clean, conductive surface with minimal resistance to electron transfer. The R\u003csub\u003ect\u003c/sub\u003e increases to 22 Ω after the first antibody attachment. After the gelatin blocking and antigen binding, further resistance increases are observed as gelatin and antigen layers add steric hindrance. The R\u003csub\u003ect\u003c/sub\u003e for the secondary antibody immobilization is about 141 Ω, reflecting the cumulative insulating effect of the immunocomplex layers. Post-substrate addition of TMB/H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, results in R\u003csub\u003ect\u003c/sub\u003e plummets to 20 Ω, nearly matching the bare electrode\u0026rsquo;s resistance. This dramatic reduction occurs because TMB⁺, generated by HRP-catalyzed oxidation, acts as a redox mediator, restoring efficient electron transfer.\u003c/p\u003e\n\u003cp\u003eThe EIS results complement the CV and DPV data, demonstrating how the biosensor\u0026rsquo;s resistance dynamically responds to bio-recognition events and enzymatic reactions. This triad of electrochemical techniques collectively validates the sensor\u0026rsquo;s design, enabling femtogram-level sensitivity and specificity for TBRFV detection. It should be mentioned that the trend of our findings in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e align well with previous reports in the literature [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe CV measurements in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea, indicated that the anodic peak current of the bare electrode surface was 705 \u0026micro;A, which decreased to 560 \u0026micro;A upon TBRFV-CP-IgG immobilization. This current further declined progressively with each step of sandwich immunocomplex formation. However, upon the addition of the enzymatic substrate, HRP catalyzed the reaction, reversing the declining trend. The peak current increased from 510 \u0026micro;A at the TBRFV-CP-IgG\u003csup\u003eHRP\u003c/sup\u003e stage to 627 \u0026micro;A, which was higher than the peak current observed after TBRFV-CP-IgG immobilization and nearly restored to the initial peak current value (705 \u0026micro;A) of the bare electrode before antibody immobilization (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e\n\u003cp\u003eSimilarly, DPV results in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb demonstrated a decrease in peak current density from 234 \u0026micro;A/cm\u0026sup2; in the bare electrode state to 205 \u0026micro;A/cm\u0026sup2; after TBRFV-CP-IgG immobilization, further declining to 150 \u0026micro;A/cm\u0026sup2; under addition of rp-CP-TBRFV as the antigen, and to 140 \u0026micro;A/cm\u0026sup2; upon TBRFV-CP-IgG\u003csup\u003eHRP\u003c/sup\u003e binding. Consistent with the CV results, exposure to TMB/H₂O₂ led to an increase in current, reaching 220 \u0026micro;A/cm\u0026sup2;, which was again higher than the TBRFV-CP-IgG immobilization stage and close to the bare electrode current density.\u003c/p\u003e\n\u003cp\u003eEIS measurements were also conducted to assess the surface resistance of the FTO electrode, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec. The resistance increased from 22 Ω after TBRFV-CP-IgG immobilization to 141 Ω upon TBRFV-CP-IgG\u003csup\u003eHRP\u003c/sup\u003e attachment. This incremental resistance trend was observed at each stage of sandwich immunocomplex formation. However, after the addition of TMB/H₂O₂, the resistance increase was halted, and the value reverted to 20 Ω.\u003c/p\u003e\n\u003cp\u003eThe observed decrease in current (CV and DPV) and increase in resistance (EIS) upon TBRFV-CP-IgG attachment, relative to the bare electrode, confirmed the successful immobilization of TBRFV-CP-IgG on the electrode surface. The subsequent decline in current and rise in resistance from TBRFV-CP-IgG to TBRFV-CP-IgG\u003csup\u003eHRP\u003c/sup\u003e stages indicated successful sandwich immunocomplex formation. However, in all measurements, the introduction of TMB/H₂O₂ reversed these trends, restoring the electrode close to its initial state before antibody immobilization.\u003c/p\u003e\n\u003cp\u003eThis phenomenon is attributed to an electrochemical reaction catalyzed by HRP in the presence of TMB/H₂O₂. TMB functions as an electroactive mediator that can undergo direct reduction on the electrode surface. HRP first catalyzes the reduction of H₂O₂, leading to the oxidation of HRP (HRP\u003csub\u003eOx\u003c/sub\u003e). This oxidized HRP is subsequently reduced by the chemically oxidized TMB (TMB\u003csub\u003eRed\u003c/sub\u003e), converting HRP\u003csub\u003eOx\u003c/sub\u003e) to its reduced form (HRP\u003csub\u003eRed\u003c/sub\u003e). Meanwhile, TMB\u003csub\u003eRed\u003c/sub\u003e is regenerated to its oxidized form (TMB\u003csub\u003eOx\u003c/sub\u003e) through interaction with H₂O₂, releasing two water molecules. Finally, TMB\u003csub\u003eOx\u003c/sub\u003e undergoes electrochemical reduction on the electrode surface, enhancing the reduction current. It should be mentioned that our findings in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e align well with previous reports in the literature [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eFigure\u0026nbsp;2 provides a detailed electrochemical characterization of the developed immunosensor for detecting TBRFV using three distinct biosensing strategies: direct immunoassay, sandwich immunoassay, and enzymatic immunoassay. The figure is divided into six panels (a\u0026ndash;f), each presenting EIS responses and corresponding calibration curves across a concentration range of 0 to 10\u003csup\u003e5\u003c/sup\u003e fg/mL of the coat protein rp-CP-TBRFV.\u003c/p\u003e\n\u003cp\u003eThe EIS response for direct immunoassay are shown in Figs.\u0026nbsp;2a and 2b assessing the sensor\u0026rsquo;s performance in detecting rp-CP-TBRFV through direct antigen-antibody interactions. The \u0026Delta;R\u003csub\u003ect\u003c/sub\u003e increases progressively with antigen concentration. At 0 fg/mL (negative control), \u0026Delta;Rct\u0026thinsp;\u0026asymp;\u0026thinsp;22 Ω (baseline). At 10⁵ fg/mL, \u0026Delta;R\u003csub\u003ect\u003c/sub\u003e rises to ~\u0026thinsp;141Ω. A linear relationship is observed between \u0026Delta;R\u003csub\u003ect\u003c/sub\u003e and the logarithm of rp-CP-TBRFV concentration. The regression coefficient (R\u0026sup2;) is 0.99, indicating strong linearity and reliability. Calculating LoD using the formula LoD\u0026thinsp;=\u0026thinsp;S/3.3\u0026sigma;, where \u0026sigma; is the standard deviation of blank signals (n\u0026thinsp;=\u0026thinsp;3 replicates), and S is calibration curve slope, results in the LoD of the direct immunoassay as low as 1.14 fg/mL (equivalent to 65.14 aM).\u003c/p\u003e\n\u003cp\u003eTo enhance sensitivity, a sandwich immunoassay format is employed, as shown in Fig.\u0026nbsp;2c. The dual-antibody approach amplifies the signal, as evidenced by a steeper increase in \u0026Delta;Rct compared to the direct assay. At 10⁵ fg/mL, \u0026Delta;Rct reaches\u0026thinsp;~\u0026thinsp;210 Ω, reflecting the cumulative insulating effect of the antigen and secondary antibody layers. The calibration curve in Fig.\u0026nbsp;2d demonstrates a linear relationship (R\u0026sup2; = 0.97) between \u0026Delta;Rct and antigen concentration, with a calculated LoD of 1.06 fg/mL. This result demonstrates superior sensitivity due to the sandwich format, which enhances antigen-antibody interactions and the signal output.\u003c/p\u003e\n\u003cp\u003eThe enzymatic amplification strategy is showcased in Fig. 2e. After forming the sandwich complex, the TMB/H₂O₂ substrate is introduced. Consequently, \u0026Delta;Rct decreases dramatically, reverting from ~\u0026thinsp;210 Ω (post-secondary antibody) to ~\u0026thinsp;20 Ω (near baseline) at 10⁵ fg/mL. This reversal confirms the efficacy of enzymatic amplification in overcoming steric hindrance. Figure 2f quantifies this response, showing a linear decrease in \u0026Delta;Rct with increasing antigen concentration (R\u0026sup2; = 0.97).\u003c/p\u003e\n\u003cp\u003eFig. 3 demonstrates the practical applicability, sensitivity, and selectivity of the developed electrochemical immunosensor for detecting TBRFV in real agricultural samples. The figure comprises three panels (a, b, c), showcasing the sensor\u0026rsquo;s performance in infected leaf and seed extracts, calibration curves, and cross-reactivity tests against non-target viruses.\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea presents the electrochemical response curves for a dilution series of TBRFV-infected leaf extracts, ranging from 1:64 to 1:512 dilutions. The immunosensor employs the direct immunoassay format, where TBRFV-specific antibodies (TBRFV-CP-IgG) capture the viral antigen (rp-CP-TBRFV) in the samples. The results reveal a concentration-dependent decrease in current intensities, with detectable signals even at the highest dilution (1:512). This indicates the sensor\u0026rsquo;s ability to identify ultra-low viral concentrations in complex biological matrices. The progressive signal attenuation correlates with reduced antigen availability at higher dilutions, validating the assay\u0026rsquo;s dynamic range.\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb illustrates the corresponding calibration curve for the leaf extract dilution series. The plot exhibits a linear relationship between the electrochemical signal (\u0026Delta;R\u003csub\u003ect\u003c/sub\u003e) and the logarithm of the dilution factor, with a regression coefficient (R\u0026sup2;) of 0.99. This strong linearity confirms the sensor\u0026rsquo;s reliability and reproducibility in quantifying TBRFV across varying concentrations.\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec evaluates the immunosensor\u0026rsquo;s selectivity by testing its response to TBRFV-infected leaf and seed extracts versus negative controls and samples containing non-target viruses (Tobacco mosaic virus [TMV], Cucumber mosaic virus [CMV], Tomato yellow leaf curl virus [TYLCV]). The results show that in the leaf extracts, TBRFV is detectable at dilutions up to 1:256, confirming robust performance in complex plant matrices. While in the seed extracts, the detection is achieved at 1:2 dilutions, highlighting the sensor\u0026rsquo;s utility in screening infected seeds, a critical pathway for viral transmission. In addition, the sensor exhibits negligible cross-reactivity (\u0026lt;\u0026thinsp;10% of the TBRFV signal) with TMV, CMV, TYLCV, or virus-free samples. This specificity ensures reliable differentiation of TBRFV from phylogenetically related viruses, such as TMV (a tobamovirus like TBRFV).\u003c/p\u003e\n\u003cp\u003eTable \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e provides a comprehensive comparison of the analytical capabilities of various electrochemical sandwich immunoassays documented in the literature, highlighting the performance metrics of the developed TBRFV immunosensor in the context of existing biosensing platforms. The table illustrates the linear detection ranges, detection limits (LoD), and references for several biosensors targeting different pathogens and biomarkers, including Enterovirus 71, carcinoembryonic antigen (CEA), and plant viruses such as cucumber mosaic virus (CMV) and citrus tristeza virus (CTV). This comparison underscores the versatility of electrochemical biosensors in diverse fields, from medical diagnostics to agricultural biosecurity, while highlighting the superior sensitivity and performance of the developed TBRFV immunosensor. Notably, the TBRFV immunosensor developed in this study demonstrates a remarkable linear detection range from 10 to 105 fg/mL, with an ultra-low LoD of 1.14 fg/mL (equivalent to 65.14 aM) for the direct assay and 1.06 fg/mL (equivalent to 60.57 aM) for the sandwich assay. These values surpass the sensitivity of many previously reported biosensors, such as those for CEA (LoD of 0.2 pg/mL) and CMV (LoD of 0.1 mg/mL), underscoring the exceptional sensitivity of the proposed platform. The integration of nanoporous gold nanostructures and HRP-labeled antibodies in the sandwich assay format significantly enhances signal amplification, enabling femtogram-level detection of TBRFV. This comparison not only validates the superior performance of the developed immunosensor but also positions it as a highly competitive tool for ultrasensitive detection of plant viruses, with potential applications in agricultural diagnostics and biosecurity.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\" class=\"fr-table-selection-hover\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eComparison of analytical performance metrics for various electrochemical sandwich immunoassays reported in the literature, including linear detection ranges, LoD, and detection methods. The table encompasses biosensors targeting plant viruses (e.g., cucumber mosaic virus and citrus tristeza virus) as well as human and animal pathogens (e.g., Enterovirus 71 and carcinoembryonic antigen), highlighting the superior sensitivity and performance of the developed TBRFV immunosensor.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBiosensor substrate\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDetection method\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLinear range\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLoD\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRefs\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eITO/ AuNPs/ MUA/ EDC-NHS/ mAb/ EV71/ mAb-HRP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCA*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.1\u0026ndash; 600 ng/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.01 ng/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAnti-CEA/Ce-MoF@HA/Ag-HRP/GCE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCA\u003c/p\u003e\n \u003cp\u003eEIS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u0026ndash; 80 ng/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.2 pg/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCS-NG/GCE /Au@Ag/HRP-anti-CEA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDPV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0001\u0026ndash; 100 ng/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.05 pg/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPCE/ Anti- CMV/ CMV/ Anti-CMV-HRP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.1\u0026ndash;1.3 mg/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.1mg/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCP- CTV/ Ab\u003csub\u003e2\u003c/sub\u003e/ MB/ HRP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDPV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.95\u0026ndash; 10.0 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e fg/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.3 fg/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMNP\u0026ndash;HBsAb/HBsAg/HBsAb\u0026ndash;HRP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u0026ndash; 0.015 ng/ mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9\u0026nbsp;pg/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAb1/CEA/(DNA/(ZMPs-HRP-Ab2)n\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.008\u0026ndash;200 ng/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 pg/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAuC-HRP-anti-AFP/AFP-anti-AFP-O-MB-AuNPs/GCE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDPV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.005\u0026ndash;20 ng/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.5 pg/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAu nano-porous/MAA/EDC-NHS/TBRFV-CP-IgG/Gelatin/rp-CP-TBRFV/ TBRFV-CP-IgG\u003csup\u003eHRP\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEIS\u003c/p\u003e\n \u003cp\u003eCV\u003c/p\u003e\n \u003cp\u003eDPV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u0026ndash;10\u003csup\u003e5\u003c/sup\u003e fg/ mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDirect assay: 1.14 fg/mL\u003c/p\u003e\n \u003cp\u003eSandwich assay: 1.06 fg/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThis work\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e*: Chronoamperometry\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eIn this study, we have successfully developed an ultrasensitive electrochemical immunosensor for the detection of TBRFV, leveraging both direct and sandwich immunoassay formats. The biosensor employs nanoporous gold-modified electrodes and HRP-labeled antibodies to achieve exceptional sensitivity and specificity. The electrochemical characterization, conducted through CV, EIS, and DPV, confirmed the successful immobilization of TBRFV-specific antibodies, the formation of antigen-antibody complexes, and the enzymatic amplification of the electrochemical signal.\u003c/p\u003e\n\u003cp\u003eThe developed immunosensor demonstrated an ultra-low detection limit of 1.14 fg/mL (equivalent to 65.14 attomolar), which surpasses the sensitivity of conventional detection methods such as ELISA and fluorescent quantum dot-based assays. This remarkable sensitivity is attributed to the efficient signal amplification provided by the HRP-catalyzed redox reaction in the presence of the TMB/H₂O₂ substrate. The biosensor also exhibited excellent specificity, effectively distinguishing TBRFV from other closely related plant viruses, including TMV, CMV, and TYLCV. This high specificity ensures reliable detection in complex agricultural matrices, reducing the risk of false positives.\u003c/p\u003e\n\u003cp\u003eThe practical applicability of the immunosensor was validated through its ability to detect TBRFV in infected leaf and seed extracts, even at extreme dilution factors (1:256 for leaf extracts and 1:2 for seed extracts). This capability is crucial for early detection and containment of TBRFV outbreaks, particularly in agricultural settings where the virus can spread rapidly through contaminated seeds, tools, and human contact. The sensor\u0026apos;s robustness and reproducibility further underscore its potential as a reliable diagnostic tool for plant biosecurity.\u003c/p\u003e\n\u003cp\u003eThe integration of nanoporous gold nanostructures into the electrode design played a pivotal role in enhancing the sensor\u0026apos;s performance. The high surface area and excellent electrical conductivity of the nanoporous gold facilitated efficient antigen capture and signal amplification, while the biocompatibility and resistance to oxidation ensured the stability of the immunosensor over time. The use of HRP-labeled antibodies in the sandwich assay format further enhanced the sensitivity, allowing for the detection of trace amounts of TBRFV in complex samples.\u003c/p\u003e\n\u003cp\u003eThis study not only presents a highly sensitive and specific method for TBRFV detection but also highlights the broader potential of electrochemical immunosensors in agricultural diagnostics. The developed platform offers a rapid, cost-effective, and field-deployable solution for the early detection of plant pathogens, which is critical for preventing widespread agricultural losses. Future work will focus on integrating this sensing platform into portable electrochemical devices, enabling real-time monitoring and on-site diagnostics in agricultural settings. This advancement could significantly enhance the ability to manage and mitigate the impact of emerging plant viruses, safeguarding global food security.\u003c/p\u003e\n\u003cp\u003eIn conclusion, the proposed electrochemical immunosensor represents a significant step forward in the field of plant virus detection. Its ultra-low detection limit, high specificity, and practical applicability make it a powerful tool for the early and accurate detection of TBRFV, offering a promising solution for the prevention and control of viral outbreaks in agriculture. The success of this approach also opens new avenues for the development of similar biosensors for other plant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors’ contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN.R. carried out all experiments and wrote the original manuscript. A.M., M.S.B., M.R.S., and R.H.S. designed the study and provided essential materials. M.R. advises the project. A.M., M.S.B., M.R.S., and R.H.S. edited the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work received institutional support from Tarbiat Modares University as part of its doctoral dissertation funding program. No specific grants or financial awards were provided by external public, commercial, or non-profit funding agencies for this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics and Consent to Participate declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSalem, N., Mansour, A., Ciuffo, M., Falk, B. and Turina, M. A new tobamovirus infecting tomato crops in Jordan. Arch. 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Chem. 2020; 8: 589560.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-methods","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plme","sideBox":"Learn more about [Plant Methods](http://plantmethods.biomedcentral.com/)","snPcode":"13007","submissionUrl":"https://submission.nature.com/new-submission/13007/3","title":"Plant Methods","twitterHandle":"@PlantMethods","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"TBRFV, Electrochemical immunosensor, Horseradish peroxidase, Sandwich immunoassays","lastPublishedDoi":"10.21203/rs.3.rs-6247800/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6247800/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTomato 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-IgG\u003csup\u003eHRP\u003c/sup\u003e 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-IgG\u003csup\u003eHRP\u003c/sup\u003e as the detection antibody, and the formation of the complete sandwich complex. Upon exposure to the TMB/H₂O₂ substrate, HRP-catalyzed redox reactions generated a measurable electrochemical signal, enabling precise quantification of TBRFV. The developed biosensor exhibited a wide linear detection range from 0 to 10\u003csup\u003e5\u003c/sup\u003e fg/mL, with an ultra-low detection limit of 1.14 fg/mL, corresponding to 65.14 aM. Furthermore, the sensor demonstrated high specificity for TBRFV, effectively distinguishing it from potential interfering agents. The proposed electrochemical immunosensing strategy provides a highly promising platform for the early and accurate detection of TBRFV, offering significant potential for agricultural biosecurity and the prevention of viral outbreaks.\u003c/p\u003e","manuscriptTitle":"Attomolar Electrochemical Direct and Sandwich Immunoassays for the Ultrasensitive Detection of Tomato Brown Rugose Fruit Virus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-21 11:12:22","doi":"10.21203/rs.3.rs-6247800/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-02T03:04:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-28T03:50:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"331635963994850548418926924316204396250","date":"2025-04-27T14:12:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-21T17:10:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"278537979616966969643611015239869211108","date":"2025-04-02T10:37:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-31T07:14:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-24T13:16:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-24T13:15:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Methods","date":"2025-03-17T21:57:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-methods","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plme","sideBox":"Learn more about [Plant Methods](http://plantmethods.biomedcentral.com/)","snPcode":"13007","submissionUrl":"https://submission.nature.com/new-submission/13007/3","title":"Plant Methods","twitterHandle":"@PlantMethods","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3e8ce15b-88d5-44ae-9c5c-7b4233ea67e6","owner":[],"postedDate":"April 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-07T16:08:56+00:00","versionOfRecord":{"articleIdentity":"rs-6247800","link":"https://doi.org/10.1186/s13007-025-01407-3","journal":{"identity":"plant-methods","isVorOnly":false,"title":"Plant Methods"},"publishedOn":"2025-07-01 15:57:01","publishedOnDateReadable":"July 1st, 2025"},"versionCreatedAt":"2025-04-21 11:12:22","video":"","vorDoi":"10.1186/s13007-025-01407-3","vorDoiUrl":"https://doi.org/10.1186/s13007-025-01407-3","workflowStages":[]},"version":"v1","identity":"rs-6247800","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6247800","identity":"rs-6247800","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}