Regenerable fuel cells based on enzymatic redox nanoparticles – NANOFUELCELL

Synthesis of PCL-b-ßCD and PCL-b-MH block copolymers was realized using click chemistry approach. For PCL-b-ßCD, firstly, the commercially available ?-hydroxy-terminated PCL was reacted with 4-bromobutyryl chloride in CH2Cl2 in the presence of triethyleneamine to yield bromo-terminated PCL, which was further treated with excess of NaN3 in DMF to obtained Azido-functionalized-PCL (PCL-N3). Secondly, commercially obtained 6-Mono-O-(p-toluenesulfonyl)-ß-cyclodextrin (Ts-ßCD) was treated with propargylamine in DMF at 80°C for 24h to obtained acetylene-functionalized ßCD. The PCL-b- ßCD was synthesized by click reaction between PCL-N3 and acetylene-functionalized ßCD in DMF, in the presence of copper nanopowder at 65°C for three days.Similarly, the PCL-b-MH was obtained by the click reaction of PCL-N3 and acetylene functionalized MH, using same methodology described above for the synthesis of PCL-b-ßCD.The molecular characteristics of both PCL-b-ßCD and PCL-b-MH block copolymers are shown in the table below.
DCM: Nanoparticle formation: Organic solvent/H2O nanoprecipitation containing the copolymer and the desired redox probe; dialysis in water. Characterization by light scattering, transmission and scanning electron microscopy of these GNPs were also carried out in order to highlight the morphology and nanometric size of these nanoparticles. Electrochemical characterization (cyclic voltammetry) in an aqueous solvent of particles immobilized on surface or free in solution (surface modified by carbon nanotubes).
Buckypaper formation: FIltration under vacuum of a dispersion of CNTs in DMF, followed by air drying [NB: BPs may or may not contain redox species. Characterisation by SEM imaging and electrochemical methods.
CRNL: 3D printing of a U-shaped electrode holder module face to face.

The synthesis of the PCL-b-ßCD and PCL-b-MH block copolymers was carried out using a click-through chemistry approach. The PCL-b-ßCD was synthesized by a click reaction between PCL-N3 and ßCD-C=C, in the presence of copper nanopowder. Following the same strategy, the PCL-b-MH was obtained by click reaction of the PCL-N3 and maltohepatitis (MH-C=C). (Table 1).
New GNPs were made from a mixture, in different proportions, of two amphiphilic copolymers: polystyrene-block-ß-cyclodextrin (PS-b-ßCD) and polystyrene-block-maltoheptaose (PS-b-MH). These 2 copolymers are both made up of a hydrophobic block, PS, and sugar (ßCD or MH) as the hydrophilic part. Their self-assembly makes it possible to obtain GNPs with a polystyrene core covered by the surface sugars. Another axis is being pursued in the context of the realization of self-supporting electrodes based on NTCs and biocompatible such as buckypapers (BP). Thanks to their low thickness, BPs offer excellent diffusion of the substrates but limit in the long term the quantity of species in contact with the conductive material (low volume). Initial work has focused on the incorporation of block copolymers with pyrene and redox functions. Bioelectrodes protected by an alginate film on their surfaces showed better temporal stability both in storage and in operation. Microcavities were created by compressing 3 sheets of buckypaper, one of which was hollowed out and sandwiched. The imprisonment of a redox dye was undertaken to study the tightness of the microvolume created.
CRNL: The experimental design was submitted to the ethics committee and approved. The devices are tested in saline phosphate buffer containing the major ions present in the natural interstitial medium as well as 10g/L bovine serum albumin, 0.5 mM ascorbate and 5 mM glucose at 37°C. Dacron limits the diffusion of glucose.

Production of caprolactone-based nanoparticles (biosourced).
Modification of block copolymer chains to introduce electropolymerizable functions.
The possibility of regenerating the activity of these PEs and thus prolonging their performance/life by renewing the enzyme solution via the injection of a new enzyme solution will be studied.

These bioelectrodes will be implanted in rats in order to evaluate their performance and biocompatibility in vivo over a long period of time via periodic injection of enzyme solutions.

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1. X. Chen, F. Giroud, A. J. Gross, S. Cosnier (O) Exploiting alginate hydrogel coatings to improve buckypaper-based bioanode stability. XXV International Symposium on Bioelectrochemistry and Bioenergetics, Limerick (Ireland) May 26-30, 2019

NANOFUELCELL aims to develop a totally innovative concept of enzymatic fuel cells (EFC) based on electro-enzymatic reactions in aqueous solution confined in permeable conductive compartments acting as electrodes. Unlike conventional EFC, enzymes and redox mediators necessary for the enzyme wiring are not immobilized but diffuse freely. In order to keep the mediators and enzymes confined in the permeable compartment, we will design supramolecular redox mediator-assemblies or enzyme assemblies. The electrical wiring of enzymes in solution with electrodes forming a permeable chamber will be ensured by supramolecular redox mediator-assemblies freely diffusing in this cavity. This strategy will overcome the low operational stability of EFC.
These redox mediator and enzyme assemblies will be constructed from a new concept of functionalizable glyconanoparticles (GNPs). These GNPs will result from the self-assembly of bio-sourced block copolymers containing ß-cyclodextrin (ß-CD) moieties in water. The GNPs are considered in aqueous medium, as spheres whose size can be modulated (Ø = 50-700 nm) with ß-CD groups present in the outer layer. As a versatile platform, GNPs will allow the anchoring of a large variety of molecules by post-functionalization of ß-CD units by host-guest interactions. In particular, the ß-CD groups allow the inclusion of hydrophobic groups like pyrene, biotin or adamantane linked to redox mediators or enzyme molecules.The validity of the electro-enzymatic systems as bioanode and biocathode will be first demonstrated by placing all components in dialysis bag (ultrafiltration or microfiltration membranes). This compartment will trap enzymes and nanoparticles while allowing the permeation of substrates of enzymes, ie O2 and glucose.
In parallel to obtain bioelectrocatalytic reactions in solution in confined environments, the development of permeable conductive compartment based on the design of tissues of carbon nanotubes (buckypaper; BP) will be investigated. This compartment based on carbon nanotubes (CNTs) will act as an electrode and as a container for electroenzymatic systems. Thus, robust and permeable BP electrodes will be prepared by dispersion of CNTs and their combination with linear organic polymers containing pyrene groups for the crosslinking of CNTs by p-stacking interactions between pyrene and CNTs. The BP electrodes will be optimized in terms of mechanical stability and adequate porosity in order to create a permeable and conductive chamber containing a solution of redox GNPs and enzymes or enzyme-GNPs. The formation of a conductive compartment will be undertaken by the compression of several BPs to create a compact block containing a cavity. The compartment electrodes based on BPs will be filled as anode with glucose dehydrogenase and quinones-nanoparticles for glucose oxidation and with bilirubin oxidase and ABTS-nanoparticles as the cathode for O2 reduction. These bioelectrodes will be used for the mediated electron transfer of enzymes via the redox GNPs and combined to create a compartment based EFC.
The performance of these EFC and the possibility to regenerate the EFC activity by the renewal of the solution of enzymes by injection of a new enzymatic solution to extend the biofuel cell lifetime will be investigated.
These bioelectrodes will be implanted in rats to assess their performance, their lifetime and their biocompatibility in vivo upon an extended period and periodic reinjection of fresh enzyme solutions. Leakage of CNTs from the BP electrodes in the body will be monitored by magnetic resonance imaging. In particular, an alginate hydrogel modified with pyrene groups will be used to cover the BP conferring higher biocompatibility to these materials. The biocompatibility of the EFC will be studied by analyzing the temporal evolution of the fibrous capsule containing collagen, fused macrophages and poly-morphonuclear cells that will probably develop around the biofuel cell.