Ethics statements

The care of the rats was approved by the European Communities Council Directive Animal Care and Use Committee. The experiments were performed in accordance to their guiding principles (European Communities Council Directive L358-86/609/EEC). All protocols involving living animals were performed under license from the French Ministry of Agriculture (License number 38018 and 381141). The Committee on the Ethics of Animal Experiments of the Grenoble University (ComEth) approved the protocol under the number 103_LER-PRETA-JPA-01).

Chemicals

Commercial thin Multi-walled Carbon Nanotubes (9.5 nm diameter, purity > 95%) obtained from Nanocyl were used as received without any purification step. Glucose oxidase (GOx) from Aspergillus niger (100 U mg−1 solid), laccase from Trametes versicolor (20 U mg−1 solid) were purchased from Sigma–Aldrich and used without further purification. Cellulose membranes were purchased from Spectrumlabs: Spectra/Por® Dialysis membrane, MWCO 6–8000 g mol−1, flat width 32 mm, diameter 20.4 mm, vol/length 3.3 mL/cm.

Instrumentation

The electrochemical characterization and the biofuel cell tests were performed with an Autolab potentiostat 100 (Eco Chemie, Utrecht, The Netherlands).

Procedures to fabricate the bioelectrodes and the GBFC

The enzymes are mechanically confined into a CNT matrix by compression of a CNT/enzyme mixture as reported20, affording direct electrical wiring between the redox active center and the electrode. Furthermore, the CNT matrix ensures high conductivity and high porosity, necessary for the diffusion of substrates. The bioelectrodes used for implantation are pellets with 6 mm thickness and 5 mm in diameter. Although only laccase is used at the cathode, GOx is combined with catalase at the anode. Catalase catalyzes the decomposition of hydrogen peroxide, produced by unwired GOx and hence prevents enzyme deactivation and serves for local oxygen depletion21. The whole GBFC was then formed by connecting the bioanode and biocathode.

Miniaturized insulated wires (UBA3219, industrifil) are connected to the CNT pellets via a conductive ink (Electrodag 423SS) (see manuscript: Fig. 1A bottom). Each pellet was then wrapped in a cellulose acetate membrane (see manuscript: Fig. 1A middle). These electrodes were then inserted into a perforated silicone sleeve (inner diameter: 5 mm) (see manuscript: Fig. 1A top) and enclosed by a silicone ring. Before use, the dialysis membrane, solutions and catheters were sterilized using an autoclave. The wires were then introduced into a biocompatible silicone tubing (721048, Harvard Apparatus; 1.9 mm outside diameter) that were then filled to a length of 3 cm with medical grade silicone. Silicone was also used to cover the non-biocatalytic parts of the silicone mould (see manuscript: Fig. 1.B). Thus, no wound or trauma can be imputed to the electrical circuitry and the wires were protected from short circuits.

The bagging of the implanted cell was carried out under a laminar flow hood under sterile conditions. The silicone sleeve containing the electrodes is inserted in a 16 mm flat width dialysis bag with a 100–500 Daltons MWCO (131054 Spectrumlabs). The bag is then filled with approximately 1 mL sterile Ringer solution and closed while avoiding air bubbles. The biocompatibility of the resulting device in the rat is due to an autoclaved Dacron® sleeve, wrapped around the dialysis bag, cut to the right volume and then sutured with surgical filament (see manuscript: Fig. 1C).

Surgical implantation of the GBFC inside a rat

Male Wistar rats weighing 300–560 g were anesthetized with isoflurane under inhalational conditions. A median laparotomy was performed to insert the implant into the retroperitoneal space in left lateral position. The catheters containing the wires of the GBFC are subcutaneously tunnelled from the abdomen up to the head of the rat (see manuscript: Fig. 1D and 1E). The wires were then soldered to a female micro-connector (BL3.36Z fischer electronik) which was insulated and fixed to the skull by acrylic cement22. The GBFC was implanted in the retroperitoneal space (Fig. 1E) of the rat because the composition of the extracellular fluid in terms of glucose and oxygen is the same as in blood. The muscular abdominal wall and the skin were finally sutured separately and the animals allowed to recover from anaesthesia. After surgery, the animals received a single injection of an analgesic (Rimadyl, 5 mg kg−1, i.m.).

After implantation, the rats were left to recover 24 hours. Each day, the rats were connected to a potentiostat to measure the open circuit voltage of the implanted GBFC.

The rats were not immediately sacrificed after the performance studies in order to evaluate the biocompatibility of the implants. As required, the rats were euthanized under anaesthesia (sodium pentobarbital 50 mg kg−1, i.p.) by intra-cardiac injection of sodium pentobarbital (100 mg).

Power management of the glucose biofuel cell

Two different electronic devices have been designed to demonstrate the power management of the implanted biofuel cell. One is for powering a common medical digital thermometer (power consumption: 50 μA at 1.5 V) and another to light a LED (4.1 mA at 2.9 V). Both electronic designs are based on a low input voltage boost converter (BQ25504, Texas instruments, Dallas, Texas, USA). The global efficiency of this circuit is well-suited to these devices because it is optimized to operate within the GBFC's characteristics (input voltage: 0.3 V to 0.6 V, input current: 10 to 100 μA, output voltage: up to 3 V). For instance, the power efficiency of the voltage boost converter was around 75% for the LED demonstrator (average input voltage: 0.5 V for an average current of 70 μA and average output voltage of 2.9 V for an average current of 4.1 mA). The principle of the power management is the same for both demonstrators (thermometer and LED), whereby a capacitor is charged by the GBFC driving the boost voltage converter. When the capacitor voltage reaches a predetermined value, the stored power is released until the capacitor voltage decreases down to a determined value. The cycle is repeated as long as the device is charged by the biofuel cell. In the case of the demonstrator to power-on the thermometer, a capacitor value of 220 μF was chosen in order to keep the temperature displayed on an LCD screen for 10 s after a capacitor charge cycle of one minute. Regarding the LED demonstrator, the diode flashed for about 88 ms after 28 s, 52 s, 81 s, 115 s and 169 s.