Coreactant-free electrochemiluminescence immunosensors for on-farm diagnosis of viral infectious diseases (ECL-FARM)

Informations projet

ECL-FARM

N° de convention de subvention: 101109381

DOI

10.3030/101109381

Projet clôturé

Date de signature de la CE 26 Avril 2023

Date de début 1 Octobre 2023

Date de fin 30 Septembre 2025

Financé au titre de

• Marie Skłodowska-Curie Actions (MSCA)

Coût total

Aucune donnée

Contribution de l’UE

€ 215 534,40

Coordonné par

UNIVERSITY COLLEGE CORK - NATIONAL UNIVERSITY OF IRELAND, CORK
Ireland

Periodic Reporting for period 1 - ECL-FARM (Coreactant-free electrochemiluminescence immunosensors for on-farm diagnosis of viral infectious diseases (ECL-FARM))

Période du rapport: 2023-10-01 au 2025-09-30

Résumé du contexte et des objectifs généraux du projet

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.

Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent

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.

Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent)

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

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