Effect of Protection Polymer Coatings on the Performance of an Amperometric Galactose Biosensor in Human Plasma.

Galactose monitoring in individuals allows the prevention of harsh health conditions related to hereditary metabolic diseases like galactosemia. Current methods of galactose detection need development to obtain cheaper, more reliable, and more specific sensors. Enzyme-containing amperometric sensors based on galactose oxidase activity are a promising approach, which can be enhanced by means of their inclusion in a redox polymer coating. This strategy simultaneously allows the immobilization of the biocatalyst to the electroactive surface and hosts the electron shuttling units. An additional deposition of capping polymers prevents external interferences like ascorbic or uric acid as well as biofouling when measuring in physiological fuels. This work studies the protection effect of poly(2-methacryloyloxyethyl phosphorylcholine-co-glycidyl methacrylate (MPC) and polyvinylimidazole-polysulfostyrene (P(VI-SS)) when incorporated in the biosensor design for the detection of galactose in human plasma.

Continuous ex vivo glucose sensing in human physiological fluids using an enzymatic sensor in a vein replica.

Managing blood glucose can affect important clinical outcomes during the intraoperative phase of surgery. However, currently available instruments for glucose monitoring during surgery are few and not optimized for the specific application. Here we report an attempt to exploit an enzymatic sensor in a vein replica that could continuously monitor glucose level in an authentic human bloodstream. First, detailed investigations of the superficial venous systems of volunteers were carried out using ocular and palpating examinations, as well as advanced ultrasound measurements. Second, a tubular glucose-sensitive biosensor mimicking a venous system was designed and tested. Almost ideal linear dependence of current output on glucose concentration in phosphate buffer saline was obtained in the range 2.2-22.0 mM, whereas the dependence in human plasma was less linear. Finally, the developed biosensor was investigated in whole blood under homeostatic conditions. A specific correlation was found between the current output and glucose concentration at the initial stage of the biodevice operation. However, with time, blood coagulation during measurements negatively affected the performance of the biodevice. When the experimental results were remodeled to predict the response without the influence of blood coagulation, the sensor output closely followed the blood glucose level.

Cross-Linkable Polymer-Based Multi-layers for Protecting Electrochemical Glucose Biosensors against Uric Acid, Ascorbic Acid, and Biofouling Interferences.

The lifetime of implantable electrochemical glucose monitoring devices is limited due to the foreign body response and detrimental effects from ascorbic acid (AA) and uric acid (UA) interferents that are components of physiological media. Polymer coatings can be used to shield biosensors from these interferences and prolong their functional lifetime. This work explored several approaches to protect redox polymer-based glucose biosensors against such interferences by designing six targeted multi-layer sensor architectures. Biological interferents, like cells and proteins, and UA and AA interferents were found to have individual effects on the current density and operational stability of glucose biosensors, requiring individual protection and treatment. Protection against biofouling can be achieved using a poly(2-methacryloyloxyethyl phosphorylcholine-co-glycidyl methacrylate) (MPC) zwitterionic polymer coating. An enzyme-scavenging approach was compared to electrostatic repulsion by negatively charged polymers for protection against AA and UA interferences. A multi-layer novel polymer design (PD) system consisting of a cross-linkable negatively charged polyvinylimidazole-polysulfostyrene co-polymer inner layer and a cross-linkable MPC zwitterionic polymer outer layer showed the best protection against AA, UA, and biological interferences. The sensor protected using the novel PD shield displayed the lowest mean absolute relative difference between the glucose reading without the interferent and the reading value with the interferent present and also displayed the lowest variability in sensor readings in complex media. For sensor measurements in artificial plasma, the novel PD extends the linear range (R2 = 0.99) of the sensor from 0-10 mM for the control to 0-20 mM, shows a smaller decrease in sensitivity, and retains high current densities. The application of PD multi-target coating improves sensor performance in complex media and shows promise for use in sensors operating in real conditions.

Tethering zwitterionic polymer coatings to mediated glucose biosensor enzyme electrodes can decrease sensor foreign body response yet retain sensor sensitivity to glucose.

Foreign body response (FBR) is a major challenge that affects implantable biosensors and medical devices, including glucose biosensors, leading to a deterioration in device response over time. Polymer shields are often used to mitigate this issue. Zwitterionic polymers (ZPs) are a promising class of materials that reduce biofouling of implanted devices. A series of ZPs each containing tetherable epoxide functional groups was synthesised for application as a polymer shield for eventual application as implantable glucose biosensors. The polymer shields were initially tested for the ability to resist fibrinogen adsorption and fibroblast adhesion. All synthesised ZPs showed comparable behaviour to a commercial Lipidure ZP in resisting fibrinogen adsorption. Nafion, a common anionic shield used against electrochemical interferents, showed higher protein adsorption and comparable cell adhesion resistance as uncoated control surfaces. However, a poly(2-methacryloyloxyethyl phosphorylcholine-co-glycidyl methacrylate) (MPC)-type ZP showed similar behaviour to Lipidure, with approximately 50% reduced fibrinogen adsorption and 80% decrease in fibroblast adhesion compared to uncoated controls. An MPC-coated amperometric glucose biosensor showed comparable current density and a 1.5-fold increase in sensitivity over an uncoated control biosensor, whereas all other polymer shields tested, including Lipidure, Nafion and a poly(ethyleneglycol) polymer, resulted in lower sensitivity and current density. Collectively, these characteristics make MPC-polymer shield coatings an appealing possibility for use in implantable glucose sensors and other implanted devices with the aim of reducing FBR while maintaining sensor performance.

Self-Powered Detection of Glucose by Enzymatic Glucose/Oxygen Fuel Cells on Printed Circuit Boards.

Monitoring glucose levels in physiological fluids can help prevent severe complications associated with hypo- and hyper-glycemic events. Current glucose-monitoring systems require a three-electrode setup and a power source to function, which can hamper the system miniaturization to the patient discomfort. Enzymatic fuel cells (EFCs) offer the opportunity to develop self-powered and minimally invasive glucose sensors by eliminating the need for an external power source. Nevertheless, practical applications demand for cost-effective and mass-manufacturable EFCs compatible with integration strategies. In this study, we explore for the first time the use of gold electrodes on a printed circuit board (PCB) for the development of an EFC and demonstrate its application in saliva. To increase the specific surface area, the PCB gold-plated electrodes were modified with porous gold films. At the anode, glucose oxidase is immobilized with an osmium redox polymer that serves as an electron-transfer mediator. At the cathode, bilirubin oxidase is adsorbed onto the porous gold surface with a blocking agent that prevents parasitic reactions while maintaining the enzyme catalytic activity. The resulting EFC showed a linear response to glucose in phosphate buffer within the range 50 µM to 1 mM, with a sensitivity of 14.13 µA cm-2 mM-1. The sensor was further characterized in saliva, showing the linear range of detection of 0.75 to 2 mM, which is within the physiological range, and sensitivity of 21.5 µA cm-2 mM-1. Overall, this work demonstrates that PCBs are suitable platforms for EFCs, paving the way for the development of fully integrated systems in a seamless and miniaturized device.

Antimicrobial enzymatic biofuel cells.

A compact antibiotic delivery system based on enzymatic biofuel cells was prepared, in which ampicillin was released when discharged in the presence of glucose and O2. The release of ampicillin was effective in inhibiting the growth of bacterium Escherichia coli as confirmed by ex situ and in situ release studies in culture media.

Use of a Thermophile Desiccation-Tolerant Cyanobacterial Culture and Os Redox Polymer for the Preparation of Photocurrent Producing Anodes.

Oxygenic photosynthesis conducted by cyanobacteria has dramatically transformed the geochemistry of our planet. These organisms have colonized most habitats, including extreme environments such as the driest warm desert on Earth: the Atacama Desert. In particular, cyanobacteria highly tolerant to desiccation are of particular interest for clean energy production. These microorganisms are promising candidates for designing bioelectrodes for photocurrent generation owing to their ability to perform oxygenic photosynthesis and to withstand long periods of desiccation. Here, we present bioelectrochemical assays in which graphite electrodes were modified with the extremophile cyanobacterium Gloeocapsopsis sp. UTEXB3054 for photocurrent generation. Optimum working conditions for photocurrent generation were determined by modifying directly graphite electrode with the cyanobacterial culture (direct electron transfer), as well as using an Os polymer redox mediator (mediated electron transfer). Besides showing outstanding photocurrent production for Gloeocapsopsis sp. UTEXB3054, both in direct and mediated electron transfer, our results provide new insights into the metabolic basis of photocurrent generation and the potential applications of such an assisted bioelectrochemical system in a worldwide scenario in which clean energies are imperative for sustainable development.

Electroactive biofilms on surface functionalized anodes: The anode respiring behavior of a novel electroactive bacterium, Desulfuromonas acetexigens.

Surface chemistry is known to influence the formation, composition, and electroactivity of electron-conducting biofilms. However, understanding of the evolution of microbial composition during biofilm development and its impact on the electrochemical response is limited. Here we present voltammetric, microscopic and microbial community analysis of biofilms formed under fixed applied potential for modified graphite electrodes during early (90 h) and mature (340 h) growth phases. Electrodes modified to introduce hydrophilic groups (-NH2, -COOH and -OH) enhance early-stage biofilm formation compared to unmodified or electrodes modified with hydrophobic groups (-C2H5). In addition, early-stage films formed on hydrophilic electrodes are dominated by the gram-negative sulfur-reducing bacterium Desulfuromonas acetexigens while Geobacter sp. dominates on -C2H5 and unmodified electrodes. As biofilms mature, current generation becomes similar, and D. acetexigens dominates in all biofilms irrespective of surface chemistry. Electrochemistry of pure culture D. acetexigens biofilms reveal that this microbe is capable of forming electroactive biofilms producing considerable current density of > 9 A/m2 in a short period of potential-induced growth (~19 h following inoculation) using acetate as an electron donor. The inability of D. acetexigens biofilms to use H2 as a sole source electron donor for current generation shows promise for maximizing H2 recovery in single-chambered microbial electrolysis cell systems treating wastewaters.

An oxygen-reducing biocathode with "oxygen tanks".

Polytetrafluoroethylene submicro-rod materials, serving as micro-scaled "oxygen tanks" and binders, have been mixed into Os redox polymer-based bilirubin oxidase cathodes, leading to both enhanced limiting current density of the oxygen reduction reaction in neutral pH and operational stability over 16 hours.

Aqueous-Eutectic-in-Salt Electrolytes for High-Energy-Density Supercapacitors with an Operational Temperature Window of 100 °C, from -35 to +65 °C.

Water-in-salt (WIS) electrolytes are gaining increased interest as an alternative to conventional aqueous or organic ones. WIS electrolytes offer an interesting combination of safety, thanks to their aqueous character, and extended electrochemical stability window, thanks to the strong coordination between water molecules and ion salt. Nonetheless, cost, the tendency of salt precipitation, and sluggish ionic transfer leading to poor rate performance of devices are some intrinsic drawbacks of WIS electrolytes that yet need to be addressed for their technological implementation. It is worth noting that the absence of "free'' water molecules could also be achieved via the addition of a certain cosolvent capable of coordinating with water. This is the case of the eutectic mixture formed between DMSO and H2O with a molar ratio of 1:2 and a melting point as low as -140 °C. Interestingly, addition of salts at near-saturation conditions also resulted in an increase of the boiling point of the resulting solution. Herein, we used a eutectic mixture of DMSO and H2O for dissolution of LiTFSI in the 1.1-8.8 molality range. The resulting electrolyte (e.g., the so-called aqueous-eutectic-in-salt) exhibited excellent energy and power densities when operating in a supercapacitor cell over a wide range of extreme ambient temperatures, from as low as -35 °C to as high as +65 °C.

Improved operational stability of mediated glucose enzyme electrodes for operation in human physiological solutions.

Stability of glucose-oxidising enzyme electrodes is affected by substances in physiological solutions, hampering deployment as long-term implantable biosensors or fuel cells. The performance of Nafion over-coated enzyme electrodes, consisting of multiwalled carbon nanotubes and flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH) or glucose oxidase (GOx) crosslinked with osmium-complex based redox polymer, was compared to uncoated electrodes in presence of uric acid and artificial plasma. Nafion over-coating resulted in lower glucose oxidation current densities compared to no over-coating. The highest initial current density for Nafion over-coated electrodes in artificial plasma in 100 mM glucose was 8.0 ± 2.0 mA cm-2 for GOx electrodes with 0.5% w/v Nafion coating. These electrodes retained 83% of initial current after 12 h continuous operation in artificial plasma while similarly prepared FADGDH electrodes retained 58% signal. This is compared to retention of only 73% or 31% observed for GOx or FADGDH electrodes in artificial plasma with no Nafion membrane. Enzyme electrodes over-coated with Nafion maintain improved signal stability when tested continuously in the presence of uric acid, identified as being the main contributing substance to FADGDH enzyme electrode instability, showing promise for application to continuous use glucose-oxidising enzyme electrodes.

Multiplexed Electrochemical Cancer Diagnostics With Automated Microfluidics.

Microfluidic platforms can lead to miniaturisation, increased throughput and reduced reagent consumption, particularly when the processes are automated. Here, a programmable microcontroller is used for automation of a microfluidic platform configured to electrochemically determine the levels of 8 proteins simultaneously in complex liquid samples. The platform system is composed of a programmable Arduino microcontroller that controls inexpensive valve actuators, pump, magnetic stirrer and electronic display. The programmable microcontroller results in repeatable timing for each step in a complex assay protocol, such as sandwich immunoassays. Application of the platform is demonstrated using a multiplexed electrochemical immunoassay based on capture at the electrode surface of magnetic particles labelled with horseradish peroxidase and detection antibody. The multiplexed assay protocol is completed in less than 30 mins and results in detection of eight proteins associated with prostate cancer. The approach presented can be used to automate and simplify high-throughput screening campaigns, such as detection of multiple biomarkers in patient samples.

Glucose biosensor based on open-source wireless microfluidic potentiostat.

Wireless potentiostats capable of cyclic voltammetry and amperometry that connect to the Internet are emerging as key attributes of future point-of-care devices. This work presents an "integrated microfluidic electrochemical detector" (iMED) three-electrode multi-potentiostat designed around operational amplifiers connected to a powerful WiFi-based microcontroller as a promising alternative to more expensive and complex strategies reported in the literature. The iMED is integrated with a microfluidic system developed to be controlled by the same microcontroller. The iMED is programmed wirelessly over a standard WiFi network and all electrochemical data is uploaded to an open-source cloud-based server. A wired desktop computer is not necessary for operation or program uploading. This method of integrated microfluidic automation is simple, uses common and inexpensive materials, and is compatible with commercial sample injectors. An integrated biosensor platform contains four screen-printed carbon arrays inside 4 separate microfluidic detection chambers with Pt counter and pseudo Ag/AgCl reference electrodes in situ. The iMED is benchmarked with K3[Fe(CN)6] against a commercial potentiostat and then as a glucose biosensor using glucose-oxidising films of [Os(2,2'-bipyridine)2(polyvinylimidazole)10Cl] prepared on screen-printed electrodes with multi walled carbon nanotubes, poly(ethylene glycol) diglycidyl ether and flavin adenine dinucleotide-dependent glucose dehydrogenase. Potential application of this cost-effective wireless potentiostat approach to modern bioelectronics and point-of-care diagnosis is demonstrated by production of glucose oxidation currents, under pseudo-physiological conditions, using mediating films with lower redox potentials.

Extracellular Electron Transfer by the Gram-Positive Bacterium Enterococcus faecalis.

Extracellular electron transfer (EET) in microbial cells is essential for certain biotechnological applications and contributes to the biogeochemical cycling of elements and syntrophic microbial metabolism in complex natural environments. The Gram-positive lactic acid bacterium Enterococcus faecalis, an opportunistic human pathogen, is shown to be able to transfer electrons generated in fermentation metabolism to electrodes directly and indirectly via mediators. By exploiting E. faecalis wild-type and mutant cells, we demonstrate that reduced demethylmenaquinone in the respiratory chain in the bacterial cytoplasmic membrane is crucial for the EET. Heme proteins are not involved, and cytochrome bd oxidase activity was found to attenuate EET. These results are significant for the mechanistic understanding of EET in bacteria and for the design of microbial electrochemical systems. The basic findings infer that in dense microbial communities, such as in biofilm and in the large intestine, metabolism in E. faecalis and similar Gram-positive lactic acid bacteria might be electrically connected to other microbes. Such a transcellular electron transfer might confer syntrophic metabolism that promotes growth and other activities of bacteria in the microbiota of humans and animals.

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.

The In Vivo Potential-Regulated Protective Protein of Nitrogenase in Azotobacter vinelandii Supports Aerobic Bioelectrochemical Dinitrogen Reduction In Vitro.

Nitrogenase, the only enzyme known to be able to reduce dinitrogen (N2) to ammonia (NH3), is irreversibly damaged upon exposure to molecular oxygen (O2). Several microbes, however, are able to grow aerobically and diazotrophically (fixing N2 to grow) while containing functional nitrogenase. The obligate aerobic diazotroph, Azotobacter vinelandii, employs a multitude of protective mechanisms to preserve nitrogenase activity, including a "conformational switch" protein (FeSII, or "Shethna") that reversibly locks nitrogenase into a multicomponent protective complex upon exposure to low concentrations of O2. We demonstrate in vitro that nitrogenase can be oxidatively damaged under anoxic conditions and that the aforementioned conformational switch can protect nitrogenase from such damage, confirming that the conformational change in the protecting protein can be achieved solely by regulating the potential of its [2Fe-2S] cluster. We further demonstrate that this protective complex preserves nitrogenase activity upon exposure to air. Finally, this protective FeSII protein was incorporated into an O2-tolerant bioelectrosynthetic cell whereby NH3 was produced using air as a substrate, marking a significant step forward in overcoming the crippling limitation of nitrogenase's sensitivity toward O2.

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.

Bioelectrochemical Haber-Bosch Process: An Ammonia-Producing H2 /N2 Fuel Cell.

Nitrogenases are the only enzymes known to reduce molecular nitrogen (N2 ) to ammonia (NH3 ). By using methyl viologen (N,N'-dimethyl-4,4'-bipyridinium) to shuttle electrons to nitrogenase, N2 reduction to NH3 can be mediated at an electrode surface. The coupling of this nitrogenase cathode with a bioanode that utilizes the enzyme hydrogenase to oxidize molecular hydrogen (H2 ) results in an enzymatic fuel cell (EFC) that is able to produce NH3 from H2 and N2 while simultaneously producing an electrical current. To demonstrate this, a charge of 60 mC was passed across H2 /N2 EFCs, which resulted in the formation of 286 nmol NH3  mg-1 MoFe protein, corresponding to a Faradaic efficiency of 26.4 %.