Peter Ó Conghaile

Self-powered wireless carbohydrate/oxygen sensitive biodevice based on radio signal transmission

Here for the first time, we detail self-contained (wireless and self-powered) biodevices with wireless signal transmission. Specifically, we demonstrate the operation of self-sustained carbohydrate and oxygen sensitive biodevices, consisting of a wireless electronic unit, radio transmitter and separate sensing bioelectrodes, supplied with electrical energy from a combined multi-enzyme fuel cell generating sufficient current at required voltage to power the electronics. A carbohydrate/oxygen enzymatic fuel cell was assembled by comparing the performance of a range of different bioelectrodes followed by selection of the most suitable, stable combination. Carbohydrates (viz. lactose for the demonstration) and oxygen were also chosen as bioanalytes, being important biomarkers, to demonstrate the operation of the self-contained biosensing device, employing enzyme-modified bioelectrodes to enable the actual sensing. A wireless electronic unit, consisting of a micropotentiostat, an energy harvesting module (voltage amplifier together with a capacitor), and a radio microchip, were designed to enable the biofuel cell to be used as a power supply for managing the sensing devices and for wireless data transmission. The electronic system used required current and voltages greater than 44 µA and 0.57 V, respectively to operate; which the biofuel cell was capable of providing, when placed in a carbohydrate and oxygen containing buffer. In addition, a USB based receiver and computer software were employed for proof-of concept tests of the developed biodevices. Operation of bench-top prototypes was demonstrated in buffers containing different concentrations of the analytes, showcasing that the variation in response of both carbohydrate and oxygen biosensors could be monitored wirelessly in real-time as analyte concentrations in buffers were changed, using only an enzymatic fuel cell as a power supply.

Further Insights into the Catalytical Properties of Deglycosylated Pyranose Dehydrogenase from Agaricus meleagris Recombinantly Expressed in Pichia pastoris

The present study focuses on fragmented deglycosylated pyranose dehydrogenase (fdgPDH) from Agaricus meleagris recombinantly expressed in Pichia pastoris. Fragmented deglycosylated PDH is formed from the deglycosylated enzyme (dgPDH) when it spontaneously loses a C-terminal fragment when stored in a buffer solution at 4 °C. The remaining larger fragment has a molecular weight of ∼46 kDa and exhibits higher volumetric activity for glucose oxidation compared with the deglycosylated and glycosylated (gPDH) forms of PDH. Flow injection amperometry and cyclic voltammetry were used to assess and compare the catalytic activity of the three investigated forms of PDH, “wired” to graphite electrodes with two different osmium redox polymers: [Os(4,4′-dimethyl-2,2′-bipyridine)2(poly(vinylimidazole))10Cl]+ [Os(dmbpy)PVI] and [Os(4,4′-dimethoxy-2,2′-bipyridine)2(poly-(vinylimidazole))10Cl]+ [Os(dmobpy)PVI]. When “wired” with Os(dmbpy)PVI, the graphite electrodes modified with fdgPDH showed a pronounced increase in the current density with Jmax 13- and 6-fold higher than that observed for gPDH- and dgPDH-modified electrodes, making the fragmented enzyme extraordinarily attractive for further biotechnological applications. An easier access of the substrate to the active site and improved communication between the enzyme and mediator matrix are suggested as the two main reasons for the excellent performance of the fdgPDH when compared with that of gPDH and dgPDH. Three of the four glycosites in PDH: N75, N175, and N252 were assigned using mass spectrometry in conjunction with endoglycosidase treatment and tryptic digestion. Determination of the asparagine residues carrying carbohydrate moieties in PDH can serve as a solid background for production of recombinant enzyme lacking glycosylation.

Nanoporous Gold-Based Biofuel Cells on Contact Lenses

A lactate/O2 enzymatic biofuel cell (EBFC) was prepared as a potential power source for wearable microelectronic devices. Mechanically stable and flexible nanoporous gold (NPG) electrodes were prepared using an electrochemical dealloying method consisting of a pre-anodization process and a subsequent electrochemical cleaning step. Bioanodes were prepared by the electrodeposition of an Os polymer and Pediococcus sp. lactate oxidase onto the NPG electrode. The electrocatalytic response to lactate could be tuned by adjusting the deposition time. Bilirubin oxidase from Myrothecium verrucaria was covalently attached to a diazonium-modified NPG surface. A flexible EBFC was prepared by placing the electrodes between two commercially available contact lenses to avoid direct contact with the eye. When tested in air-equilibrated artificial tear solutions (3 mM lactate), a maximum power density of 1.7 ± 0.1 μW cm-2 and an open-circuit voltage of 380 ± 28 mV were obtained, values slightly lower than those obtained in phosphate buffer solution (2.4 ± 0.2 μW cm-2 and 455 ± 21 mV, respectively). The decrease was mainly attributed to interference from ascorbate. After 5.5 h of operation, the EBFC retained 20% of the initial power output.

An oxygen-independent and membrane-less glucose biobattery/supercapacitor hybrid device

Enzymatic biofuel cells can generate electricity directly from the chemical energy of biofuels in physiological fluids, but their power density is significantly limited by the performance of the cathode which is based on oxygen reduction for in vivo applications. An oxygen-independent and membrane-less glucose biobattery was prepared that consists of a dealloyed nanoporous gold (NPG) supported glucose dehydrogenase (GDH) bioanode, immobilised with the assistance of conductive polymer/Os redox polymer composites, and a solid-state NPG/MnO2 cathode. In a solution containing 10mM glucose, a maximum power density of 2.3µWcm-2 at 0.21V and an open circuit voltage (OCV) of 0.49V were registered as a biobattery. The potential of the discharged MnO2 could be recovered, enabling a proof-of-concept biobattery/supercapacitor hybrid device. The resulting device exhibited a stable performance for 50 cycles of self-recovery and galvanostatic discharge as a supercapacitor at 0.1mAcm-2 over a period of 25h. The device could be discharged at current densities up to 2mAcm-2 supplying a maximum instantaneous power density of 676 μW cm-2, which is 294 times higher than that from the biobattery alone. A mechanism for the recovery of the potential of the cathode, analogous to that of RuO2 (Electrochim. Acta 42(23), 3541-3552) is described.

Arylamine functionalization of carbon anodes for improved microbial electrocatalysis

Introduction of arylamine functional groups to graphite electrodes results in improved initial catalysis for acetate oxidation by microbial biofilms over that observed on unmodified anodes. Arylamine modified anodes achieve a current density of 3.4 A m−2 whilst unmodified anodes achieve only 1.3 A m−2 during the first batch feed cycle. The surface functionalization strategy provides a route to enhancing microbial bioelectrochemical systems process performance and for studying the complex mechanisms involved in such systems.

Analysis of Agaricus meleagris pyranose dehydrogenase N-glycosylation sites and performance of partially non-glycosylated enzymes

Pyranose Dehydrogenase 1 from the basidiomycete Agaricus meleagris (AmPDH1) is an oxidoreductase capable of oxidizing a broad variety of sugars. Due to this and its ability of dioxidation of substrates and no side production of hydrogen peroxide, it is studied for use in enzymatic bio-fuel cells. In-vitro deglycosylated AmPDH1 as well as knock-out mutants of the N-glycosylation sites N75 and N175, near the active site entrance, were previously shown to improve achievable current densities of graphite electrodes modified with AmPDH1 and an osmium redox polymer acting as a redox mediator, up to 10-fold. For a better understanding of the role of N-glycosylation of AmPDH1, a systematic set of N-glycosylation site mutants was investigated in this work, regarding expression efficiency, enzyme activity and stability. Furthermore, the site specific extend of N-glycosylation was compared between native and recombinant wild type AmPDH1. Knocking out the site N252 prevented the attachment of significantly extended N-glycan structures as detected on polyacrylamide gel electrophoresis, but did not significantly alter enzyme performance on modified electrodes. This suggests that not the molecule size but other factors like accessibility of the active site improved performance of deglycosylated AmPDH1/osmium redox polymer modified electrodes. A fourth N-glycosylation site of AmPDH1 could be confirmed by mass spectrometry at N319, which appeared to be conserved in related fungal pyranose dehydrogenases but not in other members of the glucose-methanol-choline oxidoreductase structural family. This site was shown to be the only one that is essential for functional recombinant expression of the enzyme.