The prevailing form of bacterial infection is within the urinary tract, encompassing a wide array of bacteria that harness the urinary metabolome for their growth. Through their metabolic actions, the chemical composition of the growth medium undergoes modifications as the bacteria metabolize urine compounds, leading to the subsequent release of metabolites. These changes can indirectly indicate the existence and proliferation of bacterial organisms. Here, we investigate the use of an electronic tongue, a powerful analytical instrument based on a combination of non-selective chemical sensors with a partial specificity for data gathering combined with principal component analysis, to distinguish between infected and non-infected artificial urine samples. Three prevalent bacteria found in urinary tract infections were investigated, Escherichia coli, Klebsiella pneumoniae, and Enterococcus faecalis. Furthermore, the electronic tongue analysis was supplemented with 1H NMR spectroscopy and flow cytometry. Bacteria-specific changes in compound consumption allowed for a qualitative differentiation between artificial urine medium and bacterial growth.

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.

An electronic tongue is a powerful analytical instrument based on an array of non-selective chemical sensors with a partial specificity for data gathering and advanced pattern recognition methods for data analysis. Connecting electronic tongues with electrochemical techniques for data collection has led to various applications, mostly within sensing for food quality and environmental monitoring, but also in biomedical research for the analyses of different bioanalytes in human physiological fluids. In this paper, an electronic tongue consisting of six electrodes (viz., gold, platinum, palladium, titanium, iridium, and glassy carbon) was designed and tested in authentic (undiluted, unpretreated) human saliva samples from eight volunteers, collected before and during the COVID-19 pandemic. Investigations of 11 samples using differential pulse voltammetry and a principal component analysis allowed us to distinguish between SARS-CoV-2-free and infected authentic human saliva. This work, as a proof-of-principle demonstration, provides a new perspective for the use of electronic tongues in the field of enzyme-free electrochemical biosensing, highlighting their potential for future applications in non-invasive biomedical analyses.

Bacterial infections can affect the skin, lungs, blood, and brain, and are among the leading causes of mortality globally. Early infection detection is critical in diagnosis and treatment but is a time- and work-consuming process taking several days, creating a hitherto unmet need to develop simple, rapid, and accurate methods for bacterial detection at the point of care. The most frequent type of bacterial infection is infection of the urinary tract. Here, we present a wireless-enabled, portable, potentiometric sensor for E. coli. E. coli was chosen as a model bacterium since it is the most common cause of urinary tract infections. The sensing principle is based on reduction of Prussian blue by the metabolic activity of the bacteria, detected by monitoring the potential of the sensor, transferring the sensor signal via Bluetooth, and recording the output on a laptop or a mobile phone. In sensing of bacteria in an artificial urine medium, E. coli was detected in similar to 4 h (237 +/- 19 min; n = 4) and in less than 0.5 h (21 +/- 7 min, n = 3) using initial E. coli concentrations of similar to 10(3) and 10(5) cells mL(-1), respectively, which is under or on the limit for classification of a urinary tract infection. Detection of E. coli was also demonstrated in authentic urine samples with bacteria concentration as low as 10(4) cells mL(-1), with a similar response recorded between urine samples collected from different volunteers as well as from morning and afternoon urine samples.

AbstractIn Sweden as well as internationally the teaching and research nexus has been described as the defining charac-teristics of higher education promoting generic skills such as information analysis and critical reflection. Vertically Integrated Projects has been proposed as one educational strategy where research and teaching are linked by in-viting students to take active part in actual research projects. The strategy is well aligned to Scholarship of teaching and learning enabling the transition from a teacher-centred accepted knowledge to a student-centred perspective where students are invited as producers of knowledge. The aim of the current study was to explore students’ experiences of participation in a research-based learning activity with academia and industrial partners, designed as a qualitative explorative study using focus group interviews. Findings describe not only factors students find motivating for learning, but also their experience of being part of professional life with its benefits and challenges.

Sweat is a promising biofluid in allowing for non-invasive sampling. Here, we investigate the use of a voltammetric electronic tongue, combining different metal electrodes, for the purpose of non-invasive sample assessment, specifically focusing on sweat. A wearable electronic tongue is presented by incorporating metal electrodes on a flexible circuit board and used to non-invasively monitor sweat on the body. The data obtained from the measurements were treated by multivariate data processing. Using principal component analysis to analyze the data collected by the wearable electronic tongue enabled differentiation of sweat samples of different chemical composition, and when combined with 1H-NMR sample differentiation could be attributed to changing analyte concentrations.

Non-invasive healthcare technologies are an important part of research and development nowadays due to the low cost and convenience offered to both healthcare receivers and providers. This work overviews the recent advances in the field of non-invasive electrochemical biosensors operating in secreted human physiological fluids, viz. tears, sweat, saliva, and urine. Described electrochemical devices are based on different electrochemical techniques, viz. amperometry, coulometry, cyclic voltammetry, and impedance spectroscopy. Challenges that confront researchers in this exciting area and key requirements for biodevices are discussed. It is concluded that the field of non-invasive sensing of biomarkers in bodily fluid is highly convoluted. Nonetheless, if the drawbacks are appropriately addressed, and the pitfalls are adroitly circumvented, the approach will most certainly disrupt current clinical and self-monitoring practices.

Here we report on an entirely new kind of bioelectronic device - a conventional biosupercapacitor, which is built from copper containing redox proteins. Prior to biodevice fabrication, detailed spectroelectrochemical studies of the protein, viz. Acidithiobacillus ferrooxidcats rusticyanin, in solution and in adsorbed state, were performed, including estimation of the redox potential of the T1 site (0.62 V vs. NHE), protein midpoint potential when adsorbed on a self-assembled monolayer (0.34 V vs. NHE), as well as biocapacitance of rusticyanin modified gold electrodes (115 mu F cm(-2)). The symmetrical biosupercapacitor based on two identical gold electrodes modified with rusticyanin is able to capacitively store electricity and deliver electric power accumulated mostly in the form of biopseudocapacitance, when charged and discharged externally. When charged during Just 5 s, the biosupercapacitor with a total capacitance of about 73 mu F cm(-2) provided a maximum of 4 mu A cm(-2) peak current at 0.40 V. The biodevice, which can be charged and discharged at least 50 times without a significant loss of ability to store electric energy, had a low leakage current below 50 nA cm(-2).

In the last few years, there have been an increasing number of reports where different energy harvesters are directly combined with charge storing devices, based on dual-function electrodes able to convert and store electrical energy in the same volume. This includes (bio)fuel cells harvesting chemical energy, (bio)solar cells harvesting solar energy, tribo- and piezoelectric devices harvesting mechanical energy, and thermoelectrics harvesting thermal energy, which now have been intimately combined with batteries and electrochemical capacitors. These new types of hybrid electric devices show great promise especially for the design of self-powered electronics where an integrated hybrid power system is preferable to separated ones, capable of scavenging ambient energy and simultaneously store it and in this way increasing the efficiency and enabling further miniaturization. This paper details the recent emergence of hybrid energy systems, reviewing the progress made using widely different energy harvesting techniques, which have so-far not been described in a single body of work.

This article reviews recent progress in the development of biosupercapacitors - supercapacitors fabricated using biological materials. In conventional biosupercapacitors the biomaterial serves as the pseudocapacitive component, while in self-charging biodevices the biocomponent also functions as the biocatalyst. The performance characteristics of biosupercapacitors are summarized and characterized in the perspective of the broader family of electric power devices, including biodevices. Self-charging biosupercapacitors show great promise in pulse-power delivery at the milliwatt level, typically greatly exceeding the capability of free-running bio-fuel and bio-solar cells. Thus, chemical biosupercapacitors might be suitable for powering a new generation of miniaturized electronic applications, including those intended for use in medical technology, while solar biodevices might be used as highly functional, but at the same time low-cost, environmentally friendly, and technically undemanding electric power sources.

Coimmobilization of pyranose dehydrogenase as an enzyme catalyst, osmium redox polymers [Os(4,​4'-​dimethoxy-​2,​2'-​bipyridine)​2(poly(vinylimidazole)​)​10Cl]​+ or [Os(4,​4'-​dimethyl-​2,​2'-​bipyridine)​2(poly(vinylimidazole)​)​10Cl]​+ as mediators, and carbon nanotube conductive scaffolds in films on graphite electrodes provides enzyme electrodes for glucose oxidn. The recombinant enzyme and a deglycosylated form, both expressed in Pichia pastoris, are investigated and compared as biocatalysts for glucose oxidn. using flow injection amperometry and voltammetry. In the presence of 5 mM glucose in phosphate-​buffered saline (PBS) (50 mM phosphate buffer soln., pH 7.4, with 150 mM NaCl)​, higher glucose oxidn. current densities, 0.41 mA​/cm2, are obtained from enzyme electrodes contg. the deglycosylated form of the enzyme. The optimized glucose-​oxidizing anode, prepd. using deglycosylated enzyme coimmobilized with [Os(4,​4'-​dimethyl-​2,​2'-​bipyridine)​2(poly(vinylimidazole)​)​10Cl]​+ and carbon nanotubes, was coupled with an oxygen-​reducing bilirubin oxidase on gold nanoparticle dispersed on gold electrode as a biocathode to provide a membraneless fully enzymic fuel cell. A max. power d. of 275 μW​/cm2 is obtained in 5 mM glucose in PBS, the highest to date under these conditions, providing sufficient power to enable wireless transmission of a signal to a data logger. When tested in whole human blood and unstimulated human saliva max. power densities of 73 and 6 μW​/cm2 are obtained for the same fuel cell configuration, resp.

A review. Being inspired by a very recent review entitled: "Electrocatalysis and bioelectrocatalysis - Distinction without a difference" and following the general approach employed by Prof. Dr. Schuhmann, in the current work we point to the similarities and differences between oxygen electroredn. and bioelectroredn. processes. To addnl. distinguish our paper from the recent review we touch on different bioelements, such as redox proteins and living cells, but we still keep a special emphasis on oxidoreductases, biocatalysts par excellence. Moreover, we also exclusively focus on oxygen electroredn. based on direct electron transfer reactions. On the one hand, we corroborate the previously made conclusion regarding intrinsically high activity of the active sites of biol. catalysts, esp. redox enzymes, which results in mass transfer and heterogeneous electron transfer limited currents from oxygen reducing bioelectrodes. On the other hand, we disagree with the statements regarding the exceptionality of precious metal catalysts, and the notion of a huge trade-​off between high activity and stability of non-​precious metal catalysts and bioelements. We show that the activity and stability of noble metal based cathodes is very far from perfect, esp. when these electrodes operate in complex electrolytes, such as physiol. fluids, e.g. human blood.

A direct electron transfer (DET) based sulphite/oxygen biofuel cell is reported that utilises human sulphite oxidase (hSOx) and Myrothecium verrucaria bilirubin oxidase (MvBOx) and nanostructured gold electrodes. For bioanode construction, the nanostructured gold microelectrodes were further modified with 3,3′-dithiodipropionic acid di(N-hydroxysuccinimide ester) to which polyethylene imine was covalently attached. hSOx was adsorbed onto this chemically modified nanostructured electrode with high surface loading of electroactive enzyme and in presence of sulphite high anodic bioelectrocatalytic currents were generated with an onset potential of 0.05 V vs. NHE. The biocathode contained MvBOx directly adsorbed to the deposited gold nanoparticles for cathodic oxygen reduction starting at 0.71 V vs. NHE. Both enzyme electrodes were integrated to a DET-type biofuel cell. Power densities of 8 and 1 μW cm−2 were achieved at 0.15 V and 0.45 V of cell voltages, respectively, with the membrane based biodevices under aerobic conditions.

I vår moderna värd är vi väldigt beroende av elektrisk energi som vi använder för det mesta i får vardag: för att lysa upp våra hus, generera värme, driva våra datorer och mobiltelefoner och mycket mer. Produktion av elektrisk energi har dock ofta en negativ på-verkan på vår miljö. Ett alternativt sätt att producera elektrik energi är att använda sig av bränsleceller, vilka kan liknas vid öppna batterier som ständigt kan förses med nytt bränsle och således inte behöver bytas ut efter ett tag. Bränslet som används kan väljas så att dess förbrukning inte innebär någon negativ påverkan på miljön. Den här avhandlingen fokuserar sig på en viss typ av bränsleceller, där man använder sig av specifika proteiner, enzymer, för att omvandla energi från bränslet till elektrisk energi. Som bränsle kan vanligt förekommande kolhydrater, dvs. socker, samt syre användas. Socker och syre används även av vår kropp för att skapa energi, och genom att använda sig av rätt sorts enzymer kan även bränsleceller använda sig av dessa ämnen för att producera elektrisk energi. Således är det möjligt för dessa bränsleceller att producera elektrisk energi placerade inuti oss. Dessa biobränsleceller kan sedan användas för att driva t.ex. olika sensorer direkt i vår kropp som kontinuerligt skulle kunna ge information till sjukvården, utan att använda sig av batterier som behöver bytas ut. Avhandlingen är baserad på att undersöka hur olika bränsleceller som använder sig av enzym fungerar samt att testa dem i olika mänskliga kroppsvätskor samt även inuti levande organismer. Genom att öka förståelsen för detta är förhoppningen att bränsleceller baserade på enzym inom en inte allt för avlägsen framtid kan finna tillämpningar som elektriska försörjare för t.ex. självförsörjande biomedicinska sensorer.

We present data on operation of a miniature membrane-less, direct electron transfer based enzymatic fuel cell in human sweat and saliva. The enzymatic fuel cell was fabricated following our previous reports on miniature biofuel cells, utilizing gold nanoparticle modified gold microwires with immobilized cellobiose dehydrogenase and bilirubin oxidase. The following average characteristics of miniature glucose/oxygen biodevices operating in human sweat and saliva, respectively, were registered: 580 and 560 mV open-circuit voltage, 0.26 and 0.1 μW cm–2 power density at a cell voltage of 0.5 V, with up to ten times higher power output at 0.2 V. When saliva collected after meal ingestion was used, roughly a two-fold increase in power output was obtained, with a further two-fold increase by addition of 500 μM glucose. Likewise, the power generated in sweat at 0.5 V increased two-fold by addition of 500 μM glucose.

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 sep. sensing bioelectrodes, supplied with elec. energy from a combined multi-​enzyme fuel cell generating sufficient current at required voltage to power the electronics. A carbohydrate​/oxygen enzymic 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, resp. to operate; which the biofuel cell was capable of providing, when placed in a carbohydrate and oxygen contg. buffer. In addn., 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 contg. different concns. of the analytes, showcasing that the variation in response of both carbohydrate and oxygen biosensors could be monitored wirelessly in real-​time as analyte concns. in buffers were changed, using only an enzymic fuel cell as a power supply.

Miniature, self-contained biodevices powered by biofuel cells may enable a new generation of implantable, wireless, minimally invasive neural interfaces for neurophysiological in vivo studies and for clinical applications. Here we report on the fabrication of a direct electron transfer based glucose/oxygen enzymatic fuel cell (EFC) from genuinely three-dimensional (3D) nanostructured microscale gold electrodes, modified with suitable biocatalysts. We show that the process underlying the simple fabrication method of 3D nanostructured electrodes is based on an electrochemically driven transformation of physically deposited gold nanoparticles. We experimentally demonstrate that mediator-, cofactor-, and membrane-less EFCs do operate in cerebrospinal fluid and in the brain of a rat, producing amounts of electrical power sufficient to drive a self-contained biodevice, viz. 7 μW cm−2 in vitro and 2 μW cm−2 in vivo at an operating voltage of 0.4 V. Last but not least, we also demonstrate an inductive coupling between 3D nanobioelectrodes and living neurons.

A review. Interdisciplinary research has combined the efforts of many scientists and engineers to gain an understanding of biotic and abiotic electrochem. processes, materials properties, biomedical, and engineering approaches for the development of alternative power-​generating and​/or energy-​harvesting devices, aiming to solve health-​related issues and to improve the quality of human life. This review intends to recapitulate the principles of biofuel cell development and the progress over the years, thanks to the contribution of cross-​disciplinary researchers that have combined knowledge and innovative ideas to the field. The emergence of biofuel cells, as a response to the demand of elec. power devices that can operate under physiol. conditions, are reviewed. Implantable biofuel cells operating inside living organisms have been envisioned for over fifty years, but few reports of implanted devices have existed up until very recently. The very first report of an implanted biofuel cell (implanted in a grape) was published only in 2003 by Adam Heller and his coworkers. This work was a result of earlier scientific efforts of this group to "wire" enzymes to the electrode surface. The last couple of years have, however, seen a multitude of biofuel cells being implanted and operating in different living organisms, including mammals. Herein, the evolution of the biofuel concept, the understanding and employment of catalyst and biocatalyst processes to mimic biol. processes, are explored. These potentially green technol. biodevices are designed to be applied for biomedical applications to power nano- and microelectronic devices, drug delivery systems, biosensors, and many more.

High-​redox potential laccases are powerful biocatalysts with a wide range of applications in biotechnol. We have converted a thermostable laccase from a white-​rot fungus into a blood tolerant laccase. Adapting the fitness of this laccase to the specific compn. of human blood (above neutral pH, high chloride concn.) required several generations of directed evolution in a surrogate complex blood medium. Our evolved laccase was tested in both human plasma and blood, displaying catalytic activity while retaining a high redox potential at the T1 copper site. Mutations introduced in the second coordination sphere of the T1 site shifted the pH activity profile and drastically reduced the inhibitory effect of chloride. This proof of concept that laccases can be adapted to function in extreme conditions opens an array of opportunities for implantable nanobiodevices, chem. syntheses, and detoxification.

A microscale membrane-​less biofuel cell, capable of generating elec. energy from human lachrymal liq., was developed by using the ascorbate and oxygen naturally present in tears as fuel and oxidant. The biodevice is based on three-​dimensional nanostructured gold electrodes covered with abiotic (conductive org. complex) and biol. (redox enzyme) materials functioning as efficient anodic and cathodic catalysts, resp. Three-​dimensional nanostructured electrodes were fabricated by modifying 100 μm gold wires with 17 nm gold nanoparticles, which were further modified with tetrathiafulvalene-​tetracyanoquinodimethane conducting complex to create the anode and with Myrothecium verrucaria bilirubin oxidase to create the biocathode. When operated in human tears, the biodevice exhibited the following characteristics: an open circuit voltage of 0.54 V, a maximal power d. of 3.1 μW cm-​2 at 0.25 V and 0.72 μW cm-​2 at 0.4 V, with a stable c.d. output of over 0.55 μA cm-​2 at 0.4 V for 6 h of continuous operation. These findings support the authors' proposition that an ascorbate​/oxygen biofuel cell could be a suitable power source for glucose-​sensing contact lenses to be used for continuous health monitoring by diabetes patients.

After initial testing and optimization of anode biocatalysts, a membraneless glucose​/oxygen enzymic biofuel cell possessing high coulombic efficiency and power output was fabricated and characterized. Two sugar oxidizing enzymes, namely, pyranose dehydrogenase from Agaricus meleagris (AmPDH) and flavodehydrogenase domains of various cellobiose dehydrogenases (DHCDH) were tested during the pre-​screening. The enzymes were mixed, wired and entrapped in a low-​potential Os-​complex-​modified redox-​polymer hydrogel immobilized on graphite. This anode was used in combination with a cathode based on bilirubin oxidase from Myrothecium verrucaria adsorbed on graphite. Optimization showed that the c.d. for the mixed enzyme electrode could be further improved by using a genetically engineered variant of the non-​glycosylated flavodehydrogenase domain of cellobiose dehydrogenase from Corynascus thermophilus expressed in E. coli (ngDHCtCDHC310Y) with a high glucose-​turnover rate in combination with an Os-​complex-​modified redox polymer with a high concn. of Os complexes as well as a low-​d. graphite electrode. The optimized biofuel cell with the AmPDH​/ngDHCtCDHC310Y anode showed not only a similar max. voltage as with the biofuel cell based only on the ngDHCtCDHC310Y anode (0.55 V) but also a substantially improved max. power output (20 μW​/cm2) at 300 mV cell voltage in air-​satd. physiol. buffer. Most importantly, the estd. half-​life of the mixed biofuel cell can reach up to 12 h, which is apparently longer than that of a biofuel cell in which the bioanode is based on only one single enzyme.

During directed evolution to functionally express the high redox potential laccase from the PM1 basidiomycete in Saccharomyces cerevisiae, the characteristic max. absorption at the T1 copper site (Abs610T1Cu) was quenched, switching the typical blue color of the enzyme to yellow. To det. the mol. basis of this color change, we characterized the original wild-​type laccase and its evolved mutant. Peptide printing and MALDI-​TOF anal. confirmed the absence of contaminating protein traces that could mask the Abs610T1Cu, while conservation of the redox potential at the T1 site was demonstrated by spectroelectrochem. redox titrns. Both wild-​type and evolved laccases were capable of oxidizing a broad range of substrates (ABTS, guaiacol, DMP, synapic acid) and they displayed similar catalytic efficiencies. The laccase mutant could only oxidize high redox potential dyes (Poly R-​478, Reactive Black 5, Azure B) in the presence of exogenous mediators, indicating that the yellow enzyme behaves like a blue laccase. The main consequence of over-​expressing the mutant laccase was the generation of a six-​residue N-​terminal acidic extension, which was assocd. with the failure of the STE13 protease in the Golgi compartment giving rise to alternative processing. Removal of the N-​terminal tail had a neg. effect on laccase stability, secretion and its kinetics, although the truncated mutant remained yellow. The results of CD spectra anal. suggested that polyproline helixes were formed during the directed evolution altering spectral properties. Moreover, introducing the A461T and S426N mutations in the T1 environment during the first cycles of lab. evolution appeared to mediate the alterations to Abs610T1Cu by affecting its coordinating sphere. This laccase mutant is a valuable departure point for further protein engineering towards different fates.

Here we present unequivocal exptl. proof that microscale cofactor- and membrane-less, direct electron transfer based enzymic fuel cells do produce significant amts. of elec. energy in human lachrymal liq. (tears). 100 μm diam. gold wires, covered with 17 nm gold nanoparticles, were used to fashion three-dimensional nanostructured microelectrodes, which were biomodified with Corynascus thermophilus cellobiose dehydrogenase and Myrothecium verrucaria bilirubin oxidase as anodic and cathodic bioelements, resp. The following characteristics of miniature glucose/oxygen biodevices operating in human tears were registered: 0.57 V open-circuit voltage, about 1 μW cm-2 max. power d. at a cell voltage of 0.5 V, and more than 20 h operational half-life. Theor. calcns. regarding the max. recoverable elec. energy can be extd. from the biofuel and the biooxidant, glucose and mol. oxygen, each readily available in human lachrymal liq., fully support our belief that biofuel cells can be used as elec. power sources for so called smart contact lenses.

A review of some historical developments made in the field of enzymic fuel cells, discussing important design considerations taken when constructing mediator-, cofactor-, and membrane-less biol. fuel cells. Since the topic is rather extensive, only biol. fuel cells utilizing direct electron transfer reactions on both the anodic and cathodic sides are considered. Moreover, the performance of mostly glucose/oxygen biodevices is analyzed and compared. We also present some unpublished results on mediator-, cofactor-, and membrane-less glucose/oxygen biol. fuel cells recently designed in our group and tested in different human physiol. fluids, such as blood, plasma, saliva, and tears. Finally, further perspectives for biol. fuel cell applications are highlighted.

We report on the fabrication and characterisation of a gold-nanoparticle (AuNP)-basedmediatorlesssugar/oxygen biofuel cell (BFC) operating in neutral sugar-containing buffers and human physiological fluids, such as blood and plasma. First, Corynascus thermophilus cellobiose dehydrogenase (CtCDH) and Myrothecium verrucaria bilirubin oxidase (MvBOx), used as anodic and cathodic bioelements, respectively, were immobilised on goldelectrodesmodified with 20 nm AuNPs. Detailed characterisation and optimisation of a new CDH/AuNP-based bioanode were performed and the following fundamental parameters were obtained: (i) the redox potential of the haem-containing centre of the enzyme was measured to be 75 mV vs. NHE, (ii) the surface coverage of CtCDH was found to be 0.65 pmol cm−2 corresponding to a sub-monolayer coverage of the thiol-modified AuNPs by the enzyme, (iii) a turnover number for CtCDH immobilised on thiol-modified AuNPs was calculated to be ca. 0.5 s−1, and (iv) the maximal current densities as high as 40 μA cm−2 were registered in sugar-containing neutral buffers. Second, both biomodified electrodes, namely the CtCDH/AuNP-based bioanode and the MvBOx/AuNP-based biocathode, were combined into a functional BFC and the designed biodevices were carefully investigated. The following characteristics of the mediator-, separator- and membrane-less, miniature BFC were obtained: in phosphate buffer; an open-circuit voltage of 0.68 V, a maximum power density of 15 μW cm−2 at a cell voltage of 0.52 V and in human blood; an open-circuit voltage of 0.65 V, a maximum power density of 3 μW cm−2 at a cell voltage of 0.45 V, respectively. The estimated half-lives of the biodevices were found to be >12, <8, and <2 h in a sugar-containing buffer, human plasma, and blood, respectively. The basic characteristics of mediatorlesssugar/oxygen BFCs were significantly improved compared with previously designed biodevices, because of the usage of three-dimensional AuNP-modifiedelectrodes.

The catalytic cycle of multicopper oxidases (MCOs) involves intramol. electron transfer (IET) from the Cu-T1 copper ion, which is the primary site of the one-electron oxidns. of the substrate, to the trinuclear copper cluster (TNC), which is the site of the four-electron redn. of dioxygen to water. In this study we report a detailed characterization of the kinetic and electrochem. properties of bilirubin oxidase (BOx) - a member of the MCO family. The exptl. results strongly indicate that under certain conditions, e.g. in alk. solns., the IET can be the rate-limiting step in the BOx catalytic cycle. The data also suggest that one of the catalytically relevant intermediates (most likely characterized by an intermediate oxidn. state of the TNC) formed during the catalytic cycle of BOx has a redox potential close to 0.4 V, indicating an uphill IET process from the T1 copper site (0.7 V) to the Cu-T23. These suggestions are supported by calcns. of the IET rate, based on the exptl. obsd. Gibbs free energy change and theor. ests. of reorganization energy obtained by combined quantum and mol. mech. (QM/MM) calcns.

The influence of gold support on the bioelectrocatalytic activity of glucose oxidase and cellobiose dehydrogenase immobilized on self-assembled monolayer-modified high surface area gold electrodes such as rough gold and gold nanoparticles has been studied. The two types of enzyme-modified electrodes showed very high activity towards sugar oxidation. However, it has been shown that the largest part of this electrocatalytic activity comes from the underlying gold surface. These findings are of special importance for bioelectrochemical studies of enzymatic electrodes, where the immobilization support might show electrocatalytic properties toward the substrates of investigated enzymes.

To elucidate the mechanism of bilirubin oxidase (BOx) function in order to design efficient and stable biocathodes working at different conditions, the enzyme was studied thoroughly. BOx is a copper-containing redox enzyme that catalyzes the oxidation of a variety of different organic and inorganic compounds with concomitant reduction of O2 directly to H2O.