Periodic Reporting for period 1 - ECL-FARM (Coreactant-free electrochemiluminescence immunosensors for on-farm diagnosis of viral infectious diseases (ECL-FARM))
Período documentado: 2023-10-01 hasta 2025-09-30
The ECL-FARM project developed a portable, coreactant-free electrochemiluminescence (ECL) biosensing platform for early on-farm detection of Bovine Viral Diarrhoea Virus (BVDV), one of the most economically damaging cattle diseases. Current diagnostic methods, ELISA, virus isolation, and RT-PCR, are accurate but slow, laboratory-based, and unsuitable for field use.
ECL technology offers exceptional sensitivity for detecting disease biomarkers; however, commercial ECL systems are large, lab-based instruments designed for human clinical testing and depend on high concentrations of coreactants (e.g. tripropylamine (toxic)) or hydrogen peroxide (unstable) to generate light.
ECL-FARM overcomes these limitations by introducing a coreactant-free ECL platform that produces reactive oxygen species (ROSs) in situ to trigger luminol emission without the need for added coreactant chemicals. The platfrom is a miniaturised, multiplexed, and coreactant-free and label-free biosensor capable of detecting biomarkers at concentrations below 1 fg·mL⁻¹.
The system integrates silicon microfabrication, nanomaterial-based biointerfaces, a peptide antifouling layer, and a low-power photomultiplier detector, forming a compact, field-ready diagnostic tool. This innovation supports the EU Green Deal, Farm to Fork Strategy, and One Health framework by enabling sustainable, rapid, and high-sensitivity animal health monitoring.
ECL technology offers exceptional sensitivity for detecting disease biomarkers; however, commercial ECL systems are large, lab-based instruments designed for human clinical testing and depend on high concentrations of coreactants (e.g. tripropylamine (toxic)) or hydrogen peroxide (unstable) to generate light.
ECL-FARM overcomes these limitations by introducing a coreactant-free ECL platform that produces reactive oxygen species (ROSs) in situ to trigger luminol emission without the need for added coreactant chemicals. The platfrom is a miniaturised, multiplexed, and coreactant-free and label-free biosensor capable of detecting biomarkers at concentrations below 1 fg·mL⁻¹.
The system integrates silicon microfabrication, nanomaterial-based biointerfaces, a peptide antifouling layer, and a low-power photomultiplier detector, forming a compact, field-ready diagnostic tool. This innovation supports the EU Green Deal, Farm to Fork Strategy, and One Health framework by enabling sustainable, rapid, and high-sensitivity animal health monitoring.
The project successfully developed a compact miniaturised multiplexed silicon-chip ECL platform featuring entwined spiral microelectrodes in a generator–collector configuration. Seven electrode geometries were designed and fabricated using photolithography, achieving gaps and widths as small as 2 µm and ensuring crack-free, defect-free, and reproducible chip production.
A custom 3D-printed cell holder and a miniature, low-power photomultiplier tube (PMT) were integrated into the setup, resulting in a fully functional, portable detection system.
Using luminol, coreactant-free ECL was achieved through in situ generation of reactive oxygen species (ROSs) at the generator electrode. This innovation produced an 11-fold stronger light signal than single-electrode systems, demonstrating high efficiency and stability without harmful chemicals on a compact, multi-modal microelectrode platform.
For biosensing, the electrodes were coated with a chitosan nanocomposite hydrogel, then functionalised with Protein A/G to introduce oriented immobilisation for anti-IgG antibodies. A custom-designed branched peptide was incorporated to prevent nonspecific adsorption from biological fluids. The resulting biosensor achieved ultrasensitive detection limits below 1 fg·mL⁻¹, outperforming existing portable methods.
Building on this proof of concept, the platform was further adapted for BVD antibody biomarkers detection. A custom-designed peptide antifouling layer was developed and covalently attached to the electrode surface to prevent nonspecific protein adsorption, maintaining stable electrochemical performance even in 100% serum. Using this optimised surface, ultrasensitive detection of BVD antibodies was achieved, with detection limits below 1 fg·mL⁻¹ in buffer and strong potential demonstrated for application in whole serum. These results confirm the platform’s robustness and its suitability for early, on-site disease detection in complex biological samples.
A portable electronic controller was also developed, allowing electrochemical operation and data collection through a Bluetooth smartphone interface, further advancing the technology toward on-farm diagnostics.
A custom 3D-printed cell holder and a miniature, low-power photomultiplier tube (PMT) were integrated into the setup, resulting in a fully functional, portable detection system.
Using luminol, coreactant-free ECL was achieved through in situ generation of reactive oxygen species (ROSs) at the generator electrode. This innovation produced an 11-fold stronger light signal than single-electrode systems, demonstrating high efficiency and stability without harmful chemicals on a compact, multi-modal microelectrode platform.
For biosensing, the electrodes were coated with a chitosan nanocomposite hydrogel, then functionalised with Protein A/G to introduce oriented immobilisation for anti-IgG antibodies. A custom-designed branched peptide was incorporated to prevent nonspecific adsorption from biological fluids. The resulting biosensor achieved ultrasensitive detection limits below 1 fg·mL⁻¹, outperforming existing portable methods.
Building on this proof of concept, the platform was further adapted for BVD antibody biomarkers detection. A custom-designed peptide antifouling layer was developed and covalently attached to the electrode surface to prevent nonspecific protein adsorption, maintaining stable electrochemical performance even in 100% serum. Using this optimised surface, ultrasensitive detection of BVD antibodies was achieved, with detection limits below 1 fg·mL⁻¹ in buffer and strong potential demonstrated for application in whole serum. These results confirm the platform’s robustness and its suitability for early, on-site disease detection in complex biological samples.
A portable electronic controller was also developed, allowing electrochemical operation and data collection through a Bluetooth smartphone interface, further advancing the technology toward on-farm diagnostics.
ECL-FARM achieved several breakthrough innovations that advance ECL biosensing far beyond existing laboratory-based technologies:
1-Coreactant-free ECL emission: The platform eliminates the need for toxic or unstable chemical coreactants, enabling a greener, simpler, and more sustainable detection process.
2-Novel microelectrode architecture: This is the first demonstration of entwined spiral microelectrodes in ECL, achieving up to 93% redox cycling efficiency and providing enhanced signal strength and stability within a compact, miniaturised format.
3-Portable instrumentation: A low-power photomultiplier tube (PMT) integrated with a smartphone-controlled electronic interface enables real-time, on-farm electrochemical and ECL measurements with minimal energy consumption.
4-Advanced antifouling biointerface: A custom-designed peptide layer was developed to prevent nonspecific adsorption from complex biological fluids. Two biosensing configurations were established:
Configuration 1: The peptide was applied as a blocking layer on a Protein A/G–functionalised surface, enabling oriented antibody immobilisation for model IgG detection. This configuration acts as a universal immunosensing layer suitable for any IgG isotype antibody.
Configuration 2: The peptide was covalently attached to the chitosan nanocomposite hydrogel to form a fully antifouling surface, supporting direct immobilisation of BVDV Erns protein for specific antibody detection.
In both configurations, the peptide outperformed conventional blocking agents (e.g. BSA, SuperBlock), maintaining over 90% signal retention in 25% bovine fetal serum (BFS) and excellent antifouling performance in 100% serum, with stable ECL and electrochemical behaviour and resistance to nonspecific protein adsorption.
Ultrasensitive and specific immunosensing: Detection limits below 1 fg·mL⁻¹ were achieved for model IgG targets, representing one of the highest sensitivities reported for portable biosensing systems. The peptide-based platform was successfully adapted for BVD antibody and BVDV-1 Erns protein detection, showing sub-femtogram sensitivity in buffer and strong potential for direct detection in whole serum.
Together, these innovations establish a new generation of reagent-free, miniaturised, and portable immunobiosensors for early, on-site, and highly sensitive diagnostic applications, directly supporting EU objectives under the Green Deal, Farm-to-Fork Strategy, and One Health framework by promoting sustainable, rapid, and accessible animal health monitoring technologies
1-Coreactant-free ECL emission: The platform eliminates the need for toxic or unstable chemical coreactants, enabling a greener, simpler, and more sustainable detection process.
2-Novel microelectrode architecture: This is the first demonstration of entwined spiral microelectrodes in ECL, achieving up to 93% redox cycling efficiency and providing enhanced signal strength and stability within a compact, miniaturised format.
3-Portable instrumentation: A low-power photomultiplier tube (PMT) integrated with a smartphone-controlled electronic interface enables real-time, on-farm electrochemical and ECL measurements with minimal energy consumption.
4-Advanced antifouling biointerface: A custom-designed peptide layer was developed to prevent nonspecific adsorption from complex biological fluids. Two biosensing configurations were established:
Configuration 1: The peptide was applied as a blocking layer on a Protein A/G–functionalised surface, enabling oriented antibody immobilisation for model IgG detection. This configuration acts as a universal immunosensing layer suitable for any IgG isotype antibody.
Configuration 2: The peptide was covalently attached to the chitosan nanocomposite hydrogel to form a fully antifouling surface, supporting direct immobilisation of BVDV Erns protein for specific antibody detection.
In both configurations, the peptide outperformed conventional blocking agents (e.g. BSA, SuperBlock), maintaining over 90% signal retention in 25% bovine fetal serum (BFS) and excellent antifouling performance in 100% serum, with stable ECL and electrochemical behaviour and resistance to nonspecific protein adsorption.
Ultrasensitive and specific immunosensing: Detection limits below 1 fg·mL⁻¹ were achieved for model IgG targets, representing one of the highest sensitivities reported for portable biosensing systems. The peptide-based platform was successfully adapted for BVD antibody and BVDV-1 Erns protein detection, showing sub-femtogram sensitivity in buffer and strong potential for direct detection in whole serum.
Together, these innovations establish a new generation of reagent-free, miniaturised, and portable immunobiosensors for early, on-site, and highly sensitive diagnostic applications, directly supporting EU objectives under the Green Deal, Farm-to-Fork Strategy, and One Health framework by promoting sustainable, rapid, and accessible animal health monitoring technologies