ABSTRACT. The high surface areas of nanostructured electrodes can provide for significantly enhanced surface loadings of electroactive materials. The fabrication and characterisation of nanoporous gold (np-Au) substrates as electrodes for bioelectrochemical applications is described.Robust np-Au electrodes were prepared by sputtering a gold-silver alloy onto a glass support and subsequent de-alloying of the silver component. Alloy layers were prepared with either a uniform or non-uniform distribution of silver and, post de-alloying, showed clear differences in morphology on characterisation with scanning electron microscopy. Redox reactions under kinetic control, in particular measurement of the charge required to strip a gold oxide layer, provided the most accurate measurements of the total electrochemically addressable electrode surface area, A real .Values of A real up to 28 times that of the geometric electrode surface area, A geo , were obtained. For diffusion controlled reactions overlapping diffusion zones between adjacent nanopores established limiting semi-infinite linear diffusion fields where the maximum current density was dependent on A geo . The importance of measuring the surface area available for the immobilisation was determined 2 using the redox protein, cyt c. The area accessible to modification by a biological macromolecule, A macro , such as cyt c was reduced by up to 40 % compared to A real , demonstrating that the confines of some nanopores were inaccessible to large macromolecules due to steric hindrances. Preliminary studies on the preparation of np-Au electrodes modified with osmium redox polymer hydrogels and Myrothecium verrucaria bilirubin oxidase (MvBOD) as a biocathode were performed; current densities of 500 A cm -2 were obtained in unstirred solutions.

The enzyme Trametes hirsuta laccase undergoes direct electron transfer at unmodified nanoporous gold electrodes, displaying a current density of 28 A/cm 2 . The response indicates that ThLc was immobilised at the surface of the nanopores in a manner which promoted direct electron transfer, in contrast to the absence of a response at unmodified polycrystalline gold electrodes. The bioelectrocatalytic activity of ThLc modified nanoporous gold electrodes was strongly dependent on the presence of halide ions. Fluoride completely inhibited the enzymatic response, whereas in the presence of 150 mM Cl -, the current was reduced to 50% of the response in the absence of Cl -. The current increased by 40% when the temperature was increased from 20°C to 37°C. The response is limited by enzymatic and/or enzyme electrode kinetics and is 30% of that observed for ThLc co-immobilised with an osmium redox polymer. Keywords: Laccase, direct electron transfer, nanoporous gold 2 IntroductionElectron transfer (ET) reactions are ubiquitous in nature. Controlling the rate of these reactions can be of significant benefit in developing biochemical systems that utilise redox proteins and enzymes and in particular, in applications that provide power for implanted or portable electronic devices. ET can be achieved directly (direct electron transfer, DET) or by the use of mediators (mediated electron transfer, MET) to shuttle electrons between the redox centre of the enzyme and the electrode. However, mediators are unselective and can also give rise to interfering effects [1][2][3]. DET-based devices offer high selectivity and sensitivity due to the absence of mediators. In addition, devices based on DET operate at potentials close to the redox potential of the enzyme, maximising the potential difference between the cathode and anode for biofuel cell applications [2]. Moreover, DET can be used to provide detailed information on the kinetics and thermodynamics of the ET process. However, the low stability of the enzyme layer together with the long distances over which ET occurs, represent major obstacles for DET based devices. In addition, the shielding mechanism of the enzymatic redox centre by the protein shell can disrupt DET [2,4]. One approach in optimizing and enabling rapid DET is to design the electrode surface with an architecture which promotes efficient rates of ET. In this way the electrode morphology ensures the most efficient orientation of the enzymatic redox centre and facilitates communication with the electroactive surface. Porous electrodes can be used to encapsulate the enzyme within a network of cavities, shortening the distance for electron transfer to the redox active site, which will promote more efficient and rapid rates of electron transfer [5]. However, the number of redox enzymes capable of interacting directly with the electrode while catalyzing the enzymatic reaction is limited, with estimates of approximately 5% of all enzymes exhibiting such a response [6].DET in enzymes was first described in 1978 fo...

Well defined mediatorless bioelectrocatalytic reduction of oxygen with high current densities of 0.8 mA cm -2 were obtained on nanoporous gold electrodes modified with Myrothecium verrucaria bilirubin oxidase. A stable faradaic response was observed when the enzyme modified electrode was coated with a specifically designed electrodeposition polymer layer.The response of the enzyme electrode was only slightly inhibited by the addition of F -.

Nanoporous gold (NPG) fabricated by sputtering is a material of versatile morphology with pores whose size can be tailored to accommodate enzymes. The process of pore formation and the size of the pores in NPG are influenced by the composition of Au and Ag in the alloy used to prepare the electrodes together with the temperature and time period of the dealloying process. On increasing the time from 1 to 60 min and the temperature from 0.5 °C to 60.5 °C in concentrated HNO3, significant increases in the average pore diameters from 4.4 to 78 nm were observed with simultaneous decreases in the roughness factor (Rf). The pores of NPG were fully addressable regardless of the diameter, with Rf increasing linearly up to an alloy thickness of 500 nm. The influence of the pore size on the bioelectrochemical response of redox proteins was evaluated using cytochrome c as a model system. The highest current densities of ca. 30 µA cm−2 were observed at cytochrome c modified NPG electrodes with an average pore size of ca. 10 nm. The pores in NPG were also tuned for the mediatorless immobilization of Myrothecium verrucaria bilirubin oxidase. High current densities of ca. 65 µA cm−2 were observed at MvBOD modified NPG electrodes prepared by dealloying at 0.5 °C for 5 min with an average pore size of 8 nm, which is too small to accommodate the enzyme into the pores, indicating that the response was from enzyme adsorbed on the electrode surface.

Nanoporous gold (NPG) electrodes were prepared by dealloying sputtered gold:silver alloys. Electrodes of different thicknesses and pore sizes areas were prepared by varying the temperature and duration of the dealloying procedure; these were then used as supports for FAD‐dependent glucose dehydrogenase (GDH) (Glomorella cingulata) and bilirubin oxidase (BOx) (Myrothecium verrucaria). Glucose dehydrogenase was immobilized by drop‐casting a solution of the enzyme with an osmium redox polymer together with a crosslinked polymer, whereas bilirubin oxidase was attached covalently through carbodiimide coupling to a diazonium‐modified NPG electrode. The stability of the bilirubin‐oxidase‐modified NPG electrode was significantly improved in comparison with that of a planar gold electrode. Enzyme fuel cells were also prepared; the optimal response was obtained with a BOx‐modified NPG cathode (500 nm thickness) and a GDH‐modified anode (300 nm), which generated power densities of 17.5 and 7.0 μW cm−2 in phosphate‐buffered saline and artificial serum, respectively.

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.

Nanoporous gold electrodes were utilized as a support for the detection of d‐fructose. The immobilization of fructose dehydrogenase was achieved by adsorption on thiol‐ and diazonium‐bound carboxylic acid functional groups and subsequent crosslinking through cabodiimide‐initiated amide bond formation. The biosensor showed a linear response in the range 0.05–0.3 mM of d‐fructose with a sensitivity of 3.7±0.2 μA cm−2 mM−1 and a limit of detection of 1.2 μM. The response of the biosensor in a range of natural sweeteners and beverages compared very favorably to the results obtained by a commercially available kit. Accurate readings were obtainable after a very fast response time of less than 5 seconds. The biosensor showed high specificity towards d‐fructose in the presence of interfering sugars.

Abstract:Nanoporous and planar gold electrodes were utilised as supports for the redox enzymes Aspergillus niger glucose oxidase (GOx) and Corynascus thermophilus cellobiose dehydrogenase (CtCDH). Electrodes modified with hydrogels containing enzyme, Os-redox polymers and the cross-linking agent poly(ethylene glycol)diglycidyl ether (PEGDGE) were used as biosensors for the determination of glucose and lactose. Limits of detection of 6.0 (± 0.4), 16.0 (± 0.1) and 2.0 (± 0.1) µM were obtained for CtCDH modified lactose and glucose biosensors and GOx modified glucose biosensors, respectively, at nanoporous gold electrodes. Biofuel cells comprised of GOx and CtCDH modified gold electrodes were utilised as anodes, together with Myrothecium verrucaria bilirubin oxidase (MvBOD) or Melanocarpus albomyces laccase (rMaLc) as cathodes, in biofuel cells. A maximum power density of 41 µW/cm 2 was obtained for a CtCDH/MvBOD biofuel cell in 5 mM lactose and O 2 saturated buffer (pH 7.4, 0.1 M phosphate, 150 mM NaCl).2