Focus on Biofuel Cells and Self-Powered Biosensors for Smart Communities

Diabetes mellitus is a disorder in which the body does not produce enough or respond normally to insulin; consequently, blood glucose levels increase to become abnormally high. Accordingly, the primary treatment of diabetes is to control glycemic levels continuously. To continuously control glycemic levels, several medical devices have been developed to monitor blood glucose levels, represented by sensors and monitors for the self-monitoring of blood glucose. The ultimate goal for those engaged in research to develop medical devices is to develop implantable biodevices, namely self-powered autonomously operated artificial pancreas systems. One of the most challenging issues in realizing an implantable artificial pancreas is the long-term continuous supply of electricity, which is currently dependent on rechargeable batteries, requiring periodical replacement. In this work, we report the development of a direct electron transfer type enzyme-based miniaturized self-powered glucose sensor based on the BioCapacitor principle with a micro-sized enzyme anode area (0.15 mm × 0.75 mm), which has only 0.1 mm² of electrode surface. As a result, a BioCapacitor utilizing a biofuel cell with a micro-sized enzyme anode was operated by self-power. In addition, the glucose concentration was detected within the range from 13 mM to 100 mM based on the frequency of charge/discharge cycles of the BioCapacitor. Although further improvement of the current density of the micro-sized anode is necessary to monitor a glucose concentration range lower than 13 mM, this self-powered glucose sensor with a micro-sized electrode based on the BioCapacitor principle was operated continuously for 6.6 h at 37 °C in 100 mM potassium phosphate buffer (pH 7.0). Our success indicates the potential to realize self-powered, autonomous, and implantable sensing modules for bio devices such as glucose-sensing systems for an artificial pancreas.

In this research, we aimed to establish a guideline for designing electron mediators suitable for biofuel cells. A redox potential simulator was fabricated by combining density functional theory calculation and experiment, allowing us to select molecules with appropriate redox potentials efficiently. Previously, mediators have been developed depending on the trials and errors; thus, our strategy will speed up the development of biofuel cells with outstanding performances.

Further increases in the current density of biofuel cells are partly limited by the deactivation of enzymes upon adsorption on hydrophobic carbon materials. A hyperthermophilic enzyme, hyperthermophilic laccase, was employed in the present study and the change in the activities and secondary structures upon adsorption on carbon black (CB) were evaluated by the oxidation rate of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) and by Fourier transform infrared spectroscopy, respectively, in comparison with the mesophilic enzymes, laccase from Trametes versicolor (denoted as mesophilic laccase), and glucose oxidase (GOx). Hyperthermophilic laccase retained its specific activities and secondary structures upon adsorption on CB compared with the other two enzymes mesophilic laccase and GOx.

Porous microneedle electrodes with pores of ∼1 µm diameter were fabricated by electroless plating of nickel followed by gold on a polymer monolith of poly(glycidyl methacrylate). The specific surface area of the fabricated electrode evaluated by the Brunauer–Emmett–Teller method was 2.559 ± 0.050 m² g⁻¹ (standard error of mean), while that of the non-porous control was <0.001 m² g⁻¹. Electrochemical glucose sensors were then fabricated by immobilizing glucose oxidase on the gold-plated microneedle electrodes. The sensitivity of the porous microneedle glucose sensor between 0 and 15 mM glucose was 22.99 ± 0.72 µA mM⁻¹, and that of the non-porous control was 3.16 ± 0.56 µA mM⁻¹. The amperometry of glucose concentration in solution was demonstrated using the fabricated electrode as a working electrode, along with an Ag/AgCl reference electrode and gold counter electrode both of which were made of microneedles. These results indicate the advantages of porous structures for electrochemical sensing with increased sensitivity.

Herein, we describe the effect of varying anions in an electrolyte solution on current generation by a redox hydrogel electrode. The electrode surface is coated with a thin film of hydrogel matrix, consisting of an osmium (Os) redox polymer including tethered Os complexes, polymer backbone, and a redox enzyme. In this case, the enzymes employed are flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH), which catalyzes glucose oxidation, and the result was compared with that reported earlier for glucose oxidase (GOx). The hydrogel matrix facilitates efficient electron transfer from glucose to the electrode via collision of the Os complexes and thus acts as a mediator. The degree of impact of anions on current generation is characteristic of the Hofmeister series. Chaotropic anions, such as nitrate and chloride, increase and decrease the catalytic current produced by FAD-GDH and GOx hydrogel electrodes, respectively. Such anions can adsorb onto the cationic region of the FAD-GDH surface and induce a negative charge, which enhances electrostatic interactions between the enzyme and the positively charged Os polymer. Kosmotropic anions, such as sulphate and phosphate increase the catalytic current due to hydrogel shrinkage, which increases the relative concentrations of both enzyme and mediator within the hydrogel architecture due to an increase in density. High-performance electrode design depends on understanding the impact of ion identity on catalytic current responses of redox hydrogel electrodes.

Electrochemical grafting is a suitable technology for fabricating electrode surfaces with new chemical functionalities whilst maintaining the bulk properties of the electrode, and electrochemical amine oxidation and diazonium salt reduction are two widely used techniques to achieve this end. Herein, we report the electrochemical reductive grafting of Azure A onto multiwalled carbon nanotube (MWCNT) electrodes for the efficient wiring of flavin adenine dinucleotide (FAD) dependent glucose dehydrogenase. The diazonium salt of Azure A is formed in situ and subsequently grafted onto the electrode surface through electrochemical reduction. The formal potential of the resultant Azure-A-modified electrode shifted to −0.05 V vs. Ag/AgCl upon radical coupling to the MWCNT electrode. Electron transfer from FAD buried in the protein shell to the electrode via Azure A was then observed in the presence of glucose in the buffer solution. This study focused on the important effect of CNT mass loading on Azure-A loading as well as bioelectrocatalytic activity and storage stability. The three-dimensional porous structure of the MWCNT electrode was determined to be favorable for the immobilization of flavin adenine dinucleotide dependent glucose dehydrogenase and efficient electron transfer via the Azure-A functionalities. The optimized 300 µg CNT-loaded modified electrode on glassy carbon (3 mm diameter) retains its initial activity for 3 d and 25% of its initial activity after 10 d. Furthermore, we show that grafted Azure A is stably immobilized on the MWCNTs for 1 month; therefore, the limiting stability factor is enzyme leaching and/or deactivation.

A biofuel cell that can generate electricity using only water is expected to be used as a new energy harvester for an emergency power supply. A new 4-series/4-parallel structured paper-substrate biofuel cell was prepared using a fuel supply paper preloaded with glucose and phosphate buffer salts. When a power generation test was conducted by supplying water to the fuel-preloaded paper, the paper-based biofuel cell produced an output approximately 90% (0.84 mW) of that obtained by supplying a phosphate buffer containing glucose as the electrolyte. The open-circuit voltage was 2.1 V, and an LED could be powered by simply supplying water to the cell without using a booster circuit.

In order to improve the performance of direct electron transfer-type electrode using multicopper oxidase (MCO), it is important to shorten the distance between the redox site of the enzyme and the electrode surface to increase electron transfer efficiency between enzyme and electrode. In this study, we focused on the mobility of the MCO from hyperthermophilic archaeon, Pyrobaculum aerophilum, immobilized onto electrode surface via an affinity tag at the MCO terminus. The mobility of the immobilized enzyme was controlled by changing the density of the immobilized enzyme on the electrode surface by altering the density of the linker for enzyme immobilization. The electrode with low density of MCO immobilized on electrode surface was improved swing ability of the enzyme. It showed 265% higher current density for electrochemical O₂ reduction than that with high density of MCO immobilized on electrode surface. Biofuel cell using a cathode with a low density of MCO immobilized on the electrode showed 160% higher power density than a biofuel cell using a cathode with a high density of MCO immobilized on the electrode.

An electrical skin patch that can be flexibly attached to the skin and activated in 30 s by adding water was developed by integrating a built-in flexible glucose/O₂ biobattery. The latter consisted of a glucose dehydrogenase (GDH)-modified anode and an iron(II) phthalocyanine (FePc)-modified cathode. The quick activation of the patch components by water addition deep inside the patch was achieved by using a flexible water-absorbing sponge containing glucose and buffer electrolyte. A patch current of about 10 μA was maintained for more than 12 h by optimizing the amount of glucose and electrolyte contained in the sponge tank. The entire patch was soft and highly flexible to conform to curved skin surfaces, owing to its thinness (<2 mm) and the flexibility of all the patch components, including the enzyme electrodes based on the carbon fabric.