Periodic Reporting for period 1 - SynAb4Toxin (Synthetic antibody (SynAb) receptors for the detection and isolation of cyclic depsipeptide (CDP) toxins in food samples)
Période du rapport: 2023-04-01 au 2025-03-31
Food safety remains a growing concern across Europe and globally, with foodborne toxins posing significant risks to public health. Among these, cyclic depsipeptide (CDP) toxins such as beauvericin (BEA), enniatin B (ENN B), and cereulide (CER) are particularly problematic due to their potent bioactivity and occurrence in various food products. Detection of these toxins is complicated by their low concentration in complex matrices, and current methods rely heavily on antibody-based assays, which present limitations in terms of stability, cost, and adaptability.
This project introduced the SynAb (Synthetic Antibody) technology—a synthetic receptor platform aimed at replacing traditional antibodies in detection systems. The objectives of the project were to design and synthesise modular receptors tailored for BEA, ENN B, and CER, to integrate these receptors into solid-phase extraction (SPE) materials, and to translate this into analytical tools such as ELISA and lateral flow assays (LFA).
The expected impact of the project lies in the development of robust, scalable receptor-based sensing platforms that can be deployed for food quality control, addressing both health and regulatory challenges. By reducing dependence on biologics and improving the robustness of detection tools, SynAb opens new possibilities for analytical chemistry and the food safety industry.
This project introduced the SynAb (Synthetic Antibody) technology—a synthetic receptor platform aimed at replacing traditional antibodies in detection systems. The objectives of the project were to design and synthesise modular receptors tailored for BEA, ENN B, and CER, to integrate these receptors into solid-phase extraction (SPE) materials, and to translate this into analytical tools such as ELISA and lateral flow assays (LFA).
The expected impact of the project lies in the development of robust, scalable receptor-based sensing platforms that can be deployed for food quality control, addressing both health and regulatory challenges. By reducing dependence on biologics and improving the robustness of detection tools, SynAb opens new possibilities for analytical chemistry and the food safety industry.
Over the course of the fellowship, significant progress was made in all major scientific objectives:
1. Synthesis of SynAb Receptors (WP1)
Eleven synthetic receptors were successfully designed and synthesised using a modular approach combining carbazole and cavity-based components. This work delivered both short and long linker derivatives to modulate selectivity and optimise binding for different toxin sizes. A second generation of receptors was also developed to further explore structure–activity relationships.
2. Determination of Binding Affinity (WP2)
Binding was first explored via fluorescence titration, where only short-linker receptors exhibited a detectable signal upon toxin binding. Subsequently, a cell viability assay (MTT) was established to assess receptor–toxin interactions via cytoprotection. Although fluorescence titration was limited, the MTT assays confirmed that long-linker receptors were more effective for CER, while short-linkers worked better for BEA and ENN B. Initial SPR development is ongoing with an industry partner.
3. Receptor Immobilisation and SPE Cartridge Development (WP3)
One receptor bearing a carboxylic acid handle was successfully immobilised on aminated silica via peptide coupling. This cartridge showed effective retention of BEA and CER in spiked food matrices and was essential to overcome matrix effects in LC-MS analysis. Though reusability was limited, the SPE step significantly improved analytical sensitivity.
4. Development of ELISA and LFA Tools (WP4)
Due to the complexity of functionalising the carbazole core with biotin or lipoic acid handles, the implementation of ELISA and LFA was delayed. However, the synthetic route was eventually optimised near the end of the project, allowing access to these functionalised receptors for future integration into assay formats. Despite delays, the synthesis of these complex receptors marked a key chemical milestone.
Collectively, the project delivered a comprehensive receptor platform, validated toxin binding mechanisms, and laid the groundwork for commercial application.
1. Synthesis of SynAb Receptors (WP1)
Eleven synthetic receptors were successfully designed and synthesised using a modular approach combining carbazole and cavity-based components. This work delivered both short and long linker derivatives to modulate selectivity and optimise binding for different toxin sizes. A second generation of receptors was also developed to further explore structure–activity relationships.
2. Determination of Binding Affinity (WP2)
Binding was first explored via fluorescence titration, where only short-linker receptors exhibited a detectable signal upon toxin binding. Subsequently, a cell viability assay (MTT) was established to assess receptor–toxin interactions via cytoprotection. Although fluorescence titration was limited, the MTT assays confirmed that long-linker receptors were more effective for CER, while short-linkers worked better for BEA and ENN B. Initial SPR development is ongoing with an industry partner.
3. Receptor Immobilisation and SPE Cartridge Development (WP3)
One receptor bearing a carboxylic acid handle was successfully immobilised on aminated silica via peptide coupling. This cartridge showed effective retention of BEA and CER in spiked food matrices and was essential to overcome matrix effects in LC-MS analysis. Though reusability was limited, the SPE step significantly improved analytical sensitivity.
4. Development of ELISA and LFA Tools (WP4)
Due to the complexity of functionalising the carbazole core with biotin or lipoic acid handles, the implementation of ELISA and LFA was delayed. However, the synthetic route was eventually optimised near the end of the project, allowing access to these functionalised receptors for future integration into assay formats. Despite delays, the synthesis of these complex receptors marked a key chemical milestone.
Collectively, the project delivered a comprehensive receptor platform, validated toxin binding mechanisms, and laid the groundwork for commercial application.
This project offers several results that go beyond the current state of the art in synthetic receptor technology and toxin analysis:
1. Modular SynAb Library Development
A full library of 15 receptors was synthesised and characterised. The modularity and synthetic accessibility of the system represent an innovation over biologics, allowing rapid customisation of binding motifs for new toxins or analytes.
2. Tunable Receptor-Linker Architecture
The study revealed for the first time a correlation between linker length and toxin size: short linkers enhanced binding of smaller toxins (BEA, ENN B), while long linkers were favourable for the bulkier CER. This insight enables rational receptor design based on analyte size.
3. SPE Cartridges Based on SynAb Receptors
A functional cartridge was developed and validated for use in food matrix clean-up. The SPE approach increased analytical clarity in LC-MS analysis and demonstrated selective retention of toxins, supporting future productisation.
4. Synthetic Access to Assay-Compatible Receptors
Although implementation in ELISA and LFA was delayed, the final stages of the project overcame longstanding synthetic hurdles, delivering receptors equipped for assay integration. These structures offer new possibilities in diagnostic platform design.
By establishing an alternative to non-existant antibodies in food toxin detection, SynAb provides foundational tools for analytical chemists, regulators, and food safety professionals.
1. Modular SynAb Library Development
A full library of 15 receptors was synthesised and characterised. The modularity and synthetic accessibility of the system represent an innovation over biologics, allowing rapid customisation of binding motifs for new toxins or analytes.
2. Tunable Receptor-Linker Architecture
The study revealed for the first time a correlation between linker length and toxin size: short linkers enhanced binding of smaller toxins (BEA, ENN B), while long linkers were favourable for the bulkier CER. This insight enables rational receptor design based on analyte size.
3. SPE Cartridges Based on SynAb Receptors
A functional cartridge was developed and validated for use in food matrix clean-up. The SPE approach increased analytical clarity in LC-MS analysis and demonstrated selective retention of toxins, supporting future productisation.
4. Synthetic Access to Assay-Compatible Receptors
Although implementation in ELISA and LFA was delayed, the final stages of the project overcame longstanding synthetic hurdles, delivering receptors equipped for assay integration. These structures offer new possibilities in diagnostic platform design.
By establishing an alternative to non-existant antibodies in food toxin detection, SynAb provides foundational tools for analytical chemists, regulators, and food safety professionals.