Nanoconfinement of enzymes in nanocage-structured electrodes for bioelectrocatalytic energy conversion – CAGEZYMES

A variety of methods were developed to elaborate metal organic frameworks (MOFs) and their composites for the construction of electrodes and bioelectrodes with specific properties including pore structures, enzyme orientation and protection functions, conductivities, and electrochemical activities. Innovative MOF (bio)electrode assembly methods were developed while structural, enzymatic (e.g. biocatalysis), electrochemical, bioelectrocatalytic, and biofuel cell properties were evaluated using a wide range of methods including electrochemistry, single-crystal and powder-x ray diffraction (pXRD), electron microscopy, BET N2 adsorption/desorption, and spectroscopic techniques (e.g. Raman, FT-IR, and UV-vis). MOFs were synthesised based on solvothermal, hydrothermal and room temperature synthesis methods. New UV-vis protocols and protein assays were developed to permit reliable quantification of encapsulated redox enzymes and molecules. An important aspect concerned characterisation studies dedicated to unravelling material stability in aqueous buffer solutions.

Oxidoreductase enzymes were incorporated in the MOFs either (i) during MOF self-assembly (in situ encapsulation / biomineralisation), (ii) after MOF assembly (post assembly), or (iii) via direct immobilisation at porous MOF electrode surfaces. This project explored the bioelectrocatalytic activity of new enzyme-MOF bioelectrodes to drive eco-friendly dioxygen reduction to water, glucose oxidation to gluconolactone, and H2O2 reduction to water. New bioelectrocatalytic inhibition protocols were also developed.

Important progress was made on the synthesis and characterisation of micro- and meso-porous MOFs and MOF-derived electrodes integrating carbon nanotubes. Solvothermal and hydrothermal synthesis methods to obtain water-stable MOFs such as zeolitic imidazolate frameworks and Fe-based MIL(100) and PCN MOFs were developed. New redox-active MOFs comprising ferrocene and quinone species for the electrical wiring of enzymes were isolated. Composite and layer-by-layer strategies were developed and important insight into aqueous solution and buffer effects on MOF stability were revealed.

We made significant progress on the assembly and characterisation of MOF/enzyme composites (WP2) for biocatalysis and bioelectrocatalysis. In one strategy, post-synthetic encapsulation of enzymes in mesoporous was explored, for example, revealing the importance of ligand-directing enzyme orientation as well as porosity (e.g. 'nanocaging') on bioelectrocatalysis, as well as structural stability and encapsulation limitations. In a second general strategy, inspired by natural biomineralisation processes, we developed methods to encapsulate enzyme and/or redox molecules via in-situ encapsulation in metal organic frameworks. For example, we successfully demonstrated a new protected electroactive MOF/peroxidase bioelectrode in which the enzyme was electrically-accessible and protected from inhibition and thermal deactivation. The process was tuned to enable the formation of crystalline or amorphous structures, which were adapted for achieving effective bioelectrocatalytic oxidation of glucose and reduction of O2 and H202. The project results showed the possibility to obtain direct, mediated and mixed electron transfer regimes, that were very sensitive to the electrode design. The inhibition studies are of particular interest, since we were able to show remarkable enzyme protection and activation effects e.g. for H202 bioelectocatalysis. The first amorphous enzymatic glucose/O2 biofuel cell was also successfully demonstrated and tested, providing advantages e.g. economic benefits, the possibility to encapsulate hydrophilic mediators, and attractive voltage stability and inhibition protection.

Carbon-derived metal organic frameworks were also developed as highly conductive porous layers to replace carbon nanotubes. Proof-of-concept enzymatic bioanodes and biocathodes were achieved via polymeric binders, and require further development. The capacitive properties were attractive vis-a-vis carbon nanotubes and were subsequently evaluated in an international collaborative study.

This project involved the participation and upskilling of numerous talented young researchers from Master's to Postdoctoral level that were recruited for doctoral and further postdoc positions in France and Germany. Research outputs include numerous theses, presentations, and publications, with several key publications still to emerge in 2025.

This project focussed on advancing the field of bioelectrocatalysis and particularly the development of enhanced catalytic electrodes and biofuel cells under challenging conditions for clean and sustainable energy. This project made important advances in the domains of nanoscale surface chemistry, MOF and MOF-enzyme synthesis, porous materials science, biology, and electrochemistry. As mentioned earlier, the methodology developed could be extended for confinement of other catalysts or entities for applications including fuel cells, sensors and reactors. Enzymatic biofuel cells with typical µW to mW power outputs are currently destined for low-power applications, for example, for the powering of electronic medical devices. Miniaturised battery recycling is ineffective and ultimately leads to problems such as environmental pollution. Socially, there is an ever-increasing demand for wearable and implantable medical devices, for example, for personal health monitoring. This CAGEZYMES project delivered new fundamental results and understanding to help prolong the lifetime and enhance the performance of bioelectrocatalytic electrodes for biofuel cells and biosensors. The results obtained with peroxidases could open up new opportunities for bioreactors, due to the possibility to achieve substrate-product conversions under classically inhibiting concentrations. The development of porous materials and sustainable catalysis for clean energy conversion in this project is aligned with the European Union climate and energy strategy including EU directives to develop clean energy for Europeans with improved use of renewable materials and energy.

2 publications to date.

Enzymes are nature's catalysts that are being increasingly exploited at electrodes for studying and controlling chemical transformations. Enzymes have advantages compared to synthetic catalysts such as exquisite specificity but they are complex and fragile. The project aim is to develop molecularly-precise nanocage structures for confinement and protection of fragile redox enzymes for bioelectrocatalysis. Methods will be developed to obtain metal organic framework (MOF) electrodes with specific pore structures and chemistry. New knowledge in MOF synthesis and breakthrough methods in MOF-electrode assembly will be developed. The project will reveal if nanocage structures can stabilise enzymes, lead to new understanding of enzyme function, and result in next generation biofuel cells for sustainable energy conversion. This 4-year project will allow the esteemed junior CNRS researcher to build his research team and lead the development of a new thematic and its sub-topics in Grenoble.