Final Report - 3D-NANOBIODEVICE (Three-dimensional nanobiostructure-based self-contained devices for biomedical application)
Executive summary:
Self-contained wireless bioelectronic devices have huge scientific and practical importance for basic science and for possible applications in medicine, high-tech industry, military, and biocomputing. The anticipation of major breakthroughs in such a range of areas has resulted in significant research focus in such devices together with a rapid growth in the number of publications and patent applications in bioelectronics. The integration of biomaterials with electronic elements, such as electrodes, chips, field-effect transistors, and piezoelectric crystals, yields hybrid bioelectronic systems that may function as Biofuel cells (BFCs), biosensors, bioelectronic circuitry, and combinations thereof. The ultimate technological goal of our project 3D-NANOBIODEVICE was to construct a hybrid bioelectronic system, viz. potentially implantable self-contained biodevices working in biomatrices of different compositions, e.g. blood, serum, plasma, saliva, extracellular fluids, and cell cultures.
This goal was successfully accomplished. Specifically, at the very end of the project two proof-of-principle wireless self-powered biodevices were fabricated, calibrated, and tested. These devices contain glucose or oxygen sensitive biosensors connected to a transmitter/operating electronic device powered by BFCs. Signals from these devices, corresponding to varying concentrations of bioanalytes, are transferred to a back-end system, e.g. a computer with an electronic transducer for storage, processing, and integration of the biological information collected by the biosensors.
The main scientific goal of the project was to enhance understanding of the fundamental principles regarding the control of electron transfer reactions between nanoparticles, nanotubes, nanofibres, Os and Ru redox complexes, as well as their assemblies confined in ordered three-dimensional (3D) microscale networks combined with different bioelements, such as glucose oxidising and oxygen reducing enzymes, in order to solve one of the main obstacles of bioelectronics, i.e. the lack of (or poor) electronic communication between the biocomponents and the electronic elements. A number of novel 3D nanobiodevices were designed and tested, such as glucose and oxygen sensitive microbiosensors, nanostructured biocathodes and bioanodes, and their combinations with electronics (operating unit containing transmitter, potentiostat, electric power storage device and transducer) into autonomous and self-contained, potentially implantable devices powered by 3D BFCs. Theoretical modelling of the response of the sensors combined with a detailed experimental characterisation of the properties of the sensors were performed in glucose containing biological matrices of different composition and pH, e.g. blood, plasma, serum, and saliva. The required long-term, operational stability of the 3D nanostructured biodevices was achieved by choosing the appropriate bioelements from already available and from novel wild-type and specifically engineered highly active, stable, and halide resistant redox enzymes, such as glucose oxidase (GOx) and Cellobiose dehydrogenase (CDH), as well as high redox potential Blue multicopper oxidases (BMCO).
The self-contained biodevices that we developed are still macroscale in size. However, their miniaturisation is possible and such miniaturised biodevices can be used for continuous glucose and oxygen monitoring in different parts of the human body, thus improving the quality of life and increasing patient safety for such chronic conditions as diabetes and methemoglobinemia. Moreover, in the long-term, wireless self-powered biodevices based on nanobiostructures might be used for neuron/nerve stimulation to compensate or alleviate the effect of human disabilities as well as to treat chronic pain, cerebral palsy, and other conditions.
Beside the scientific and technological work, significant disseminative activities were carried out by the consortium. In addition to classical dissemination roots (publications in journals, conferences, symposiums, and congresses) and novel/electronic means (web), results from 3D-NANOBIODEVICE were used in lectures, education programs (summer schools, workshops, and colloquiums), and other communication materials (newsletters, flyers, posters) transmitted via mass communication media.
Project context and objectives:
The'Three-dimensional nanobiostructure-based self-contained devices for biomedical application' (3D-NANOBIODEVICE) project has been focused on several scientific, technological, and social objectives simultaneously.
One of the main scientific objectives was to enhance the understanding of the fundamental principles for controlling electron transfer reactions between gold nanoparticles (AuNPs), carbon nanotubes, carbon post materials (CPMs), conductive nano/microporous silicone and silicate (NMPSi and NMPSiT) chips, their assemblies confined into 3D microscale networks, and different bioelements, such as glucose oxidising (GOx and CDH) and oxygen reducing (laccase (Lc) and Bilirubin oxidase (BOx)) enzymes.
Another important scientific objective of the project was to understand experimental limits and fundamental differences in the performance of 3D nanobiostructures in different biomatrices, specifically human physiological fluids and cell based in vitro platforms versus simple buffer solutions.
The main technological objective of the project was to construct potentially implantable self-contained wireless biodevices for glucose and oxygen monitoring. Novel biodevices were constructed by combination of glucose and oxygen sensitive biosensors made from 3D nanobiostructured materials, and operated by an electronic unit containing a transmitter, operating system, potentiostat, and an electric energy harvesting device.
The last but not the least objective of the project, strongly oriented towards nanotechnology and biotechnology, which have usually topped most lists of areas where the 'next-big-thing' would come from, is an intensive and fair communication with the scientific community and the public at large, in full agreement with the European Commission (EC) strategic roadmap on communicating nanotechnology.
All scientific, technological, and disseminative objectives of the project were successfully accomplished as described in detail below. It is important to emphasise that the scientific and technological objectives of 3D-NANOBIODEVICElie somewhere between these two applications. By integrating fabricated nanobiostructure based biodevices and electronic elements into functional biomedical devices with a wireless transmission feature and testing the proof-of-concept for wireless glucose and oxygen sensing in real biological fluids, 3D-NANOBIODEVICE project stepped far beyond the state of the art.
Nanowiring of appropriate redox enzymes with AuNPs, carbon nanotubes, CMPs, proper surface modifications, and use of Os and Ru redox complexes, were chosen as a major direction to solve main obstacles in the area of bioelectronics, i.e. poor electronic communication between the biocomponents and the electronic elements along with insufficient operational stability. To produce 3D bioelectrodes with superior characteristics mathematical modelling of their performance was performed and the results obtained from calculations were compared against experimentally determined parameters. From the modelling and characterisation results, optimisation of the biodevices (glucose and oxygen sensitive biosensors, bioanodes, and biocathodes) was done to increase their efficiency and stability.
Such an ultimate technological goal, as the construction of potentially implantable self-powered wireless biodevices working in biomatrices of different compositions and pHs, could be achieved only in the very end of 3D-NANOBIODEVICE. The final outcome of the project has been broken down to smaller tasks, which can be divided into seven main groups:
i) development of electronic parts of the devices, their combination into the functional transmitter/receiver system, and development of the requisite software;
ii) fabrication of stable 3D-nanostructures;
iii) production of active bioelements;
iv) design of Os or Ru containing redox polymers;
v) combination of nanostructures and redox enzymes into efficient nanostructured biomodified electrodes, which could serve as glucose and oxygen sensitive biosensors, sugar oxidising bioanodes, and oxygen reducing biocathodes;
vi) fabrication and characterisation of direct ET (DET) and mediated ET (MET) based BFCs;
vii) design and testing of self-contained biodevices for glucose and oxygen monitoring.
Indeed, these scientific and technological steps were successfully reached by the consortium as detailed below:
1.1. development of a wireless transmission system with voltage multiplayer converting 0.57 V to 2.5 V integrated with capacitive power storage;
1.2. design of a micro-potentiostat integrated with glucose and oxygen biosensors and wireless transmission system;
1.3. creation of a data transmission system with optimised data transfer and antenna for data transfer over distances of more than 3 metres;
1.4. development of optimised hardware and software components as a basis for a self-contained measurement-transmitting device.
2.Electroconducting 3D nanoarchitectures were fabricated and characterised morphologically and electrochemically:
2.1. NMPSi and NMPSiT chips of area 6x6 mm2 and of depth 10-100 micrometres;
2.2. ordered assembles of nanoparticles (OAN) with NPs sizes varying from 2 to 200 nm;
2.3. hierarchical two-generation carbon nanotube-based materials (HTGCNT) 10-100 µm deep;
2.4. CPM arrays with 6x6 mm2 areas and 10-100 µm deep structures;
2.5. mesoporous gold electrodes.
3.1.Available glucose oxidising and oxygen reducing enzymes were isolated on preparative scale (up to 500 mg), purified to homogeneity, and characterised biochemically and kinetically:
3.1.1. anodicbioelements: both GOx and CDH, > 100 mg;
3.1.1. cathodicbioelements: BMCO, viz. Lcs and BOx, > 100 mg;
3.2. newly screened and designed bioelements were produced, purified, and delivered to the consortium:
3.2.1.anodicbioelements: both GOx and CDH, > 50 mg;
3.2.2. cathodicbioelements: chloride resistance high redox potential Lcs, > 50 mg.
4. Os or Ru containing redox polymers were produced, tested, and delivered to consortium. A large library of redox polymers was also designed.
5.3D biodevices functioning as glucose and oxygen sensitive biosensors, sugar oxidising bioanodes, and oxygen reducing biocathodes, were fabricated by biomodification of 3D nanostructures with suitable bioelements.
6.1.Unoptimised DET and MET based BFCs were fabricated based on available bioelements. The biodevices were characterised in simple buffer solutions and human physiological fluids, e.g. human serum.
6.2.Optimised DET and MET based BFCs were designed based on fabricated electroconducting 3D nanoarchitectures modified with novel engineered bioelements. The performance of biodevices was tested in simple buffer solutions and human physiological fluids, e.g. human blood, plasma, serum, and saliva.
7.Two wireless self-powered biodevices for glucose and oxygen monitoring were designed and tested.In addition to technological tasks the fundamental principles of the function of electroconducting biodevices based on 3D nanobiostructures were studied and some experimental limits in their performances were clarified as described below:
a) Efficient motionless 3D nanobiostuctures based biodevices for oxygen reduction perform in mass-transfer limited regime, i.e. with maximal current densities up to 0.5 mA cm-2, whereas most CDH modified bioelectrodes were limited by the activity of redox enzymes immobilised on the electrode surface. Contrary to CDH modified electrodes, some GOx modified 3D nanobiostructures also operate in mass-transfer limited regime, i.e. with maximal current densities up to 5 mA cm-2.
b) Most of the 3D nanobiosturctures based biodevices for glucose oxidation operated at quite high potentials (up to 0.5 V vs. NHE), which is not suitable for biosensing purposes and BFC applications. Several possibilities to decrease the overpotential for sugar oxidation of CDH- and GOx modified 3D electrodes were undertaken, viz. mutagenesis of redox enzymes and appropriate choice of redox mediators using a design library of Os and Ru containing redox polymers. Contrary to CDH- and GOx modified electrodes, motionless 3D nanobiostuctures based biodevices for oxygen reduction operated at potentials very close to redox equilibrium potentials of the oxygen/water couple at certain conditions (e.g. 0.7 V vs. NHE at physiological pH values).
Another scientific objective of 3D-NANOBIODEVICE entailed the investigation of fabricated and characterised 3D-nanobiostructures in human blood, plasma, saliva, and cell based in vitro platforms vs. simple buffer solutions. Specifically:
a) Needed equipment for investigation of human physiological fluids were installed, volunteers (donors) were identified, and samples were collected. This was done in accordance with ethical issues and biomedical protocols presented in detailed project description (annex I).
b) Investigations of designed nanobiostructures in blood, plasma, serum, and saliva were performed. It was shown that 3D bioelectrocatalytic structures for oxygen electroreductionlose efficiency and stability in complex physiological fluids because of the inhibition/deactivation of redox enzymes (Lc and BOx) by different organic and inorganic substances. In addition, electrochemical oxidation of different interfering compounds, primarily ascorbate occurred on the nanostructured electrodes. Two major strategies towards overcoming the expected limitations of biocathode performance can be proposed, viz. (i) blocking of the bare nanostructured surface to disable direct contact with interfering molecules and (ii) use of another electrode material, which is inert with regard to ascorbateelectrooxidation. The latter approach is quite difficult to realise since interfering molecules, e.g. ascorbate, can be efficiently electrooxidised at quite low overpotentials on most conductive electrodes. To block ascorbate molecules from bare surfaces, negatively charged polymers can be used. However, a significant efficiency decrease of the essentially membrane based biocathode compared to membrane-less biodevices is expected, owing to mass-transfer limitations. Thus, one of the best approaches is to block nanostructured surfaces with a dense monolayer of a biocatalyst using oriented enzyme immobilisation on modified electrode surfaces, as has been already done in the case of Lc-based biocathodes (partner CSIC).
In contrast, glucose oxidising 3D-nanobiostructures can, in principle, be activated by inorganic compounds, e.g. calcium ions, as was shown by Partner ULUND. However, the concentration of these compounds in human fluids is not sufficient to enable activation, while the low substrate content of biofluids (glucose, lactose, etc.) resulted in quite inefficient performance of CDH-based nanobiostructures. One of the possible solutions to improve efficiency of bioanodes in physiological biomatrices is the use of even more efficient biocatalysts, e.g. engineered CDH with low KM and high kcat values for glucose. Another approach is to increase the coulombic efficiency of anodic biodevices. The majority of currently developed biodevices extract only two electrons from glucose or lactose molecules, while much more is really possible. For instance, complete oxidation of glucose can give 24 electrons and thus approaching complete oxidation of glucose is important scientific challenge into improving the power output from bioanodes operating in energetically poor (with low amount of biofuels, e.g. glucose) physiological fluids, e.g. human saliva.
Additional testing of biomodified electrodes was performed in cell culture conditions to investigate their biocompatibility. For this purpose, oxygen reducing and glucose oxidising biodevice were designed in cell culture plates containing a permeable membrane at the bottom. The system was created by using disposable electrode arrays, 8W2x1E (applied biophysics, USA) which were modified prior to investigation biodevices. Each array contained eight wells with two planar gold electrodes each. The construction and the performance of the biodevices were optimised for several days by monitoring of both analytes under cell culture conditions. The catalytically inactive elements of biodevices, e.g. non-biomodified nanostructured surfaces based on carbon nanotubes, did not affect attachment and growth of cells on the surfaces. However, the introduction of biocatalysts, e.g. GOx or BOx enzymes rendered the surfaces strongly cytotoxic even when the biodevices were not electrically connected. The effect of GOx can be easily explained by the generation of hydrogen peroxide at GOx modified surfaces since both substrates of the enzyme are present in the solution, glucose and oxygen. In case of BOD the result cannot be explained by BOx catalytic reaction since in the absence of electron donors (without applied potential) the enzyme does not function. Further investigations are needed to understand the cell toxicity of the nanobiostructured surfaces.
It should be emphasised that wireless self-powered 3D nanobiostructure based devices developed in the frame of the project are still macroscale. Moreover, miniaturisation of electronics is also need. However, the results of the project including proof-of-principle tests show, without a doubt, that miniature self-contained biodevices can be used for continuous glucose and oxygen monitoring in different parts of the human body, thus improving quality of life and increasing patient safety in the case of widely prevalent chronic diseases such as diabetes and methemoglobinemia. Moreover, in the long term, 3D nanobiostructure based elements will be essential for constructing devices to be used for neuron/nerve stimulations and compensation of human disabilities.
Besides scientific and technological work strong communication activities were carried out by the Consortium. In addition to classical dissemination roots (publications in journals, conferences, and workshops) and novel/electronic means (web), results from '3D-NANOBIODEVICE' were used in lectures, education programs, and other communication materials (newsletters, flyers, posters) via mass communication media.
Project results:
All the work of the consortium has been done in strict observance with the structure of annex I of the grant agreement following the project objectives and description of work. The detailed plan of the work was described in five Workpackages (WPs).
WP1 - Fabrication and characterisation of microscale 3D electrodes (AU, RUB, UL, ULUND)
One of the main aims of this WP was to produce conductive, high surface area supports for the immobilisation of enzymes for use in biosensors and BFCs. High surface area supports are essential for the successful immobilisation of enzymes at loadings that can deliver the current and power requirements of the devices needed for self-contained wireless bioelectronic devices. The main objective of this WP was to produce conducting 3D nanostructures with controlled nanodimensions, surface chemistries and surface energies/curvatures for efficient performance of enzymes in 3D nanostructures. Four approaches were planned: (i) the fabrication of carbon post materials (CPM), (ii) the formation of conductive NMPSi and NMPSiT chips, (iii) the synthesis of hierarchical two-generation carbon nanotube materials (HTGCNT) grafted onto graphite electrodes and (iv) the fabrication of ordered assembles of nanoparticles (OAN) and nanoporous gold chips (NGC). Devices using each of these approaches have been prepared and characterised as described in the following sections.
WP2 - Engineering of bioelements by rational design and directed evolution (BOKU, CSIC, NZ, INBI)
The contribution of WP2 to the project involved the production of existing glucose oxidising (anode) and oxygen reducing (cathode) enzymes, as well as new wild-type enzymes obtained by screening, and the development of protein engineered variants with improved properties. The aim of this WP is to provide the consortium with the best bioelements to design efficient and stable BFCs, as well as sensitive and selective biosensors.
As a starting point, a collection of known enzymes was produced in purified form (at ~100 mg scale) and supplied to the consortium partners for initial testing. These included a variety of CDH from myriococcumthermophilum (MtCDH) and Humicolainsolens (HiCDH), and also the well-known GOx from aspergillusniger (AnGOx), which has been used in several commercial glucose biosensors. This set of enzymes has a wide range of properties (pH, glucose activity, thermal stability) and allowed for the construction and testing of bioanodes and glucose sensitive biosensors utilising both MET and DET systems, under different reaction conditions, as well as different immobilisation strategies. For testing the construction and initial performance of biocathodes, Lcs from myceliophthorathermophila (MtLc), streptomycescoelicolor (ScLc), trameteshirsuta (ThLc), rhusvernicifera (RvLc), as well as a BOx from myrotheciumverrucaria (MvBiOx), were produced, purified, and sampled. These enzymes also cover a range of characteristics, with high-, medium- and low-redox potential enzymes, of different pH optima and susceptibility to chloride ions (a common issue with laccase-based systems). Despite the variety observed in this set of available enzymes, there were a number of limitations of the bioelements that needed to be overcome to obtain an optimal bioelectrode working under physiological conditions (pH 7.4 glucose concentration of approximately 5 mM and chloride concentration of approximately150 mM). These limitations were addressed by screening for new wild-type enzymes, as well as optimising performance of the best available enzymes by protein engineering.
The sourcing of new wild type enzymes commenced using a Prussian blue-based screening assay for novel carbohydrate oxidoreductases with improved glucose turnover at pH 7.4. This process was successfully developed and validated by the subcontractor Prof. M. Rabinovich from the A.N. Bach Institute of Biochemistry, Moscow. In the following screening of several hundred fungal cultures 15 carbohydrate oxidoreductase producing fungal species were identified 7 of which were characterised and the results published (Harreither et al., 2011; Sygmund et al., 2011a). Of these enzymes, six were CDHs and one an FAD-dependent glucose dehydrogenase (GDH). After investigating the molecular and catalytic properties MtCDH, dichomerasaubinetii CDH (DsCDH), and corynascusthermophilus CDH (CtCDH) and GDH from glomerellacingulata (GcGDH) were produced on a large scale (approximately 300 mg) and distributed to the cooperating partners for testing in combination with existing electrodes. These CDHs have a significantly improved glucose turnover at pH 7.4 compared to the previously available basidiomycetous CDHs (Harreither et al., 2012).
Further improvements of DET-anode biocatalysts were pursued by means of genetic engineering using a rational, structure-based approach. MtCDH was successfully used as template to improve the Intramolecular electron transfer (IET) between the flavodehydrogenase domain and the cytochrome domain to achieve an overall faster electron transfer from the substrate to the electrode. The main steps in this process were the comparative modelling of the enzyme's structure, docking experiments to evaluate the domain interaction, the identification of factors increasing the IET, site-directed mutagenesis, recombinant production in the expression host pichiapastoris, purification by a multi-step chromatographic procedure, steady-state and transient-state kinetic measurements, and finally the evaluation of the obtained enzyme variants. Beneficial mutations were combined and final variants carried up to seven mutations which increased the IET up to 3.5-fold. In another subtask the glucose turnover in MtCDH and CtCDH was increased by mutating the active site and a combination of certain mutations. The catalytic efficiency of both CDHs for glucose turnover was increased by a factor of 5. Additionally, the catalytic efficiency for maltose, which is an interfering substance for glucose biosensors, could be reduced 100-fold.
Another subtask involved the production of a lower redox potential form of the cytochrome domain in CDH to increase the open circuit potential of the developed BFCs. For this purpose 13 positions in the primary and secondary amino acid shell around heme b cofactor of the cytochrome domain were targeted by site-directed mutagenesis. The rationale was to (i) introduce a covalent bond to the cofactor to change the b-type heme into a c-type heme, which has a lower redox potential, or (ii) reduce the number of electron withdrawing residues around the hemestetrapyrrole ring. The obtained results are ambivalent: although several enzyme variants show a lower redox potential (50 to 60 mV vs. NHE) than the wild-type enzyme (100 mV vs. NHE) the IET is also greatly reduced and limits the electron transfer rate. By using the obtained variants, the open circuit potential of the BFC could be increased by a maximum of 50 mV, but the current output would be 10 50-fold lower.
On a parallel track, protein engineering of HiCDH was employed to improve the activity on glucose at 5 mM concentration and pH 7. Initially, molecular modelling and docking experiments were performed to obtain a structural model of the enzyme and identify suitable positions for mutagenesis. The diversity was obtained by site-directed mutagenesis, targeted primary to residues in the vicinity of the active-site, and the resulting variants were screening using two assays: cytochrome c and DCPIP as electron acceptors, as measures of DET and MET, respectively. The best hits from the variant screening were expressed in Aspergillusoryzae, purified and characterised to obtain kinetic constants. The optimisation of HiCDH resulted in variants with up to 30-fold improved activity on glucose (5 mM), at pH 7, and catalytic rates above 2 s-1 (at 5 mM glucose).
Also, some optimisation work was done to improve the dehydrogenase activity of AnGOx, as a measure to improve its activity in MET electrodes. Structure-based mutagenesis with modifications on the active site resulted in improved variants with up to 3-fold higher activity on DCPIP and up to 90% reduced oxidase activity (an unwanted competing reaction). Finally, GDH from G. cingulata (GcGDH), which has a high glucose turnover (approximately 500 s-1) at pH 7.4 was employed in electrodes developed for mediated electron transfer (MET). To increase the availability of the enzyme it was cloned and recombinantly expressed in P. pastoris and kinetically characterised (Sygmund et al., 2011b). Additionally, special mutations were introduced to make the enzyme susceptible for covalent attachment to the electrode via linkers to increase electron transfer rates.
In order to obtain a superior enzyme for the second half-reaction (reduction of oxygen), taking place at the cathode, engineering of two high-redox potential Lcs from basidiomycetes PM1 and pycnoporuscinnabarinus (PM1Lc and PcLc, respectively) was undertaken. The initial strategy comprised the replacement of the native signal peptides by the alpha factor preproleader and the joint evolution of the fusion genes to give rise to detectable secretion levels in S. cerevisiae.
The PM1Lc was subjected to 8 rounds of evolution and screening, engineering a protein mutant (OB-1) with a total laccase activity improvement of 34,000-fold over the parent type (8 mg/L of secreted protein) (Figure 23). The OB-1 mutant harbored mutations V162A, H208Y, S224G, A239P, D281E, S426N and A461T in the mature protein whereas V(10)D, N(23)K and A(87)T occurred at the preproleader. OB-1 was fairly active and stable in terms of temperature (T50 = 73 degrees of Celsius), pH profile and the presence of co-solvents. The PcLc evolution was carried out in the presence of natural and artificial redox mediators enhancing the secretion levels 40-fold along with a 14-fold increase in kcat. The evolved preproleaders were useful for the secretion of native laccases in yeast and constituted a valuable tool for further protein engineering. The evolved PcLc was also highly secreted by other heterologous host (Aspergillusniger) at levels as high as 23 mg/L. The evolved PcLcharbored mutations N208S, R280H, N331D, D341N and P394H in mature protein whereas showed A)(9)D, F(48)S, S(58)G, G(62)R and E(86)G at the preproleader. PM1Lc and PcLc evolved laccases were subjected to a chimeragenesis protocol to shuffle the properties of both enzymes finding new variants with novel pH profiles and/or improved thermal stabilities.
The second challenge of evolving these laccases was to improve the activity at neutral pH and in the presence of high concentrations of chloride ions (human blood resistant). The PM1Lc evolved for functional expression and activity (OB-1 mutant) was selected as starting point to tailor a human blood tolerant laccase. A screening protocol based on the use of surrogate blood (blood buffer with similar biochemical composition as blood but in the absence of cells and coagulation agents) was employed to stress enzymes in the presence of chlorides at alkaline pH.To make a breakdown of the evolved properties several re-screens were incorporated in each generation finding out the individual improvements in terms of inhibition (vs. halides and hydroxides) as well as stability. After four cycles of evolution and semi-rational approaches (including several site- and saturation- mutagenesis experiments in different positions of the OB-1 gene) an ultimate variant (ChU-B mutant) was engineered containing 2 mutations located at the second coordination sphere of the T1 Cu site (F396I and F454G). The evolved mutant was biochemically characterised from two glycoforms with similar features and slight differences in glycosylations degrees (with molecular weights of 60310 and 58052 Da, respectively). Regardless of the isoform, the pH profile was noticeably shifted as main consequence of the directed evolution (even changing the maximum pH for activity vs. phenolic compounds form 4.0 to 5.0). The residual activity of the ChU-B mutant at pH 7.4 (pH of blood) was 7% for ABTS and 23% for DMP. We are not aware about reports of other HRPLs showing activity at pH above 7.0. ChU-B mutant enhanced its I50 for chlorides from 169 mM up to 1024 mM. A less pronounced effect was detected for fluorides (which were not targeted for evolution) with a change in the I50 from 69 to 109 mM, for parent and ChU-B mutant respectively. In general terms, the ChU-B mutant showed turnovers in blood buffer of 427 and 143 mol product mol laccase-1 min-1, for DMP and ABTS, respectively. Neither the parent nor other HRPLs tested in our laboratory (e.g. TrameteshirsutaLc) display activity under such conditions. In collaboration with partner 7B, the mutant was preliminarily characterised in terms of electrochemical responses and direct electron transfer has been detected at electrodes in blood buffer. Finally, in collaboration with Mah, the mutant was tested in real human blood and plasma, showing activity (measured as consumption of oxygen and using ascorbic acid as reducing substrate). This last mutant was over-expressed in pichiapastoris in collaboration with BOKU and supplied to the consortium for different trials (around 25 mg of pure mutant protein was delivered to the consortium in the final meeting). The mutant was successfully tested in the prototype designed during the final workshop, which took place at Bochum, Germany (RUB).
In summary, the results from WP2 provided improved glucose-oxidising enzymes both for DET (engineered CDHs) and MET (GcGDH and engineered AnGOx), as well as oxygen-reducing enzymes, viz. laccase variants with high redox potential and activity in blood buffer, for the construction of glucose and oxygen sensitive biosensors, as well as glucose/oxygen BFCs, much superior to the available bioelements at the start of the project.
WP3 -Biomodification of the selected 3D electrodes (Mah, AU, RUB, UL, CSIC, ULUND, NUIG, US)
The main objective of WP3 is to investigate, and report on, methods for electronically addressing glucose oxidising and oxygen reducing enzymes (from WP2) in 3D structures (from WP1). The programme was split into two tasks, the first focused on synthesis and characterisation of a range of redox complexes and polymers that can mediate electron transfer between enzymes and electrodes, and the second task focused on screening approaches for modification of 3D electrode surfaces with components (enzyme, and mediator if necessary) to provide preliminary results on enzyme electrode signal, and signal stability, as a precursor to further tests on biosensor and biopower device operation in WP4.
(i) Generation and characterisation of redox polymers (RUB and NUIG)
Redox complexes, and redox polymers, are used as components of enzyme electrodes in biosensor and biopower devices to enable efficient mediated electron transfer (MET) between enzyme and electrode (electronic wiring of enzyme). A robotic synthesiser system, and/or combinatorial parallel synthesis, using a range of monomeric components and polypyridyl ligands provided for polymer and redox complex libraries. Combination, through parallel synthesis, of elements from the two libraries were then used to prepare a range of redox polymers with a diversity of redox potential, and physicochemical properties, that could be tailored for the application desired, such as redox enzyme active site potential or immobilisation approach. Preliminary characterisation of the polymers, the redox complexes and the redox polymers enabled reporting of the polymer composition (Table 1) and redox complex/polymer potential (Os2+/3+ transition).
Initial testing of enzyme electrodes prepared using co-immobilisation of redox polymers with model enzymes focused on simple physisorption onto solid (carbon) electrodes and evaluation of the bioelectrocatalytic currents for substrate oxidation (for sugar-oxidising enzymes) or reduction (for oxygenase enzymes). For example RUB have characterised the response for bioelectrocatalytic reduction of oxygen in thin films of selected redox polymers co-immobilised with the Trametes hirsute laccase as a function of enzyme loading.
NUIG investigated glucose oxidation by thin films of redox polymer co-immobilised on graphite with glucose oxidase in different ratios, using different crosslinking agents in the presence and absence of added carbon nanotubes.
(ii) Screening for various procedures for modification/activation of electrode surfaces for facilitating immobilisation/ET with biocomponents and/or mediators
Four types of 3D-nanostructures were fabricated in WP1: (i) CMP, (ii) NMPSi and NMPSiT, (iii) HTGCNT, and (iv) OAN. Work on NMPSi and NMPSiT was not pursued as these electrodes were not sufficiently stable (silicate materials) or did not provide the performance needed (silicon electrodes). These materials were replaced with porous gold modified electrodes which were shown to provide high surface areas (WP1).
Finally, as part of WP3 flexible surface attachment chemistry (US) for carbon surfaces was developed using a high-yielding maleimide coupling of enzyme to pre-treated carbon surfaces, as reported on in deliverable 3.3. WP4 - Characterisation and optimisation of microscale 3-D biodevices (AU, RUB, UL, CSIC, ULUND, NUIG, US)
The aim of WP4 represented the evaluation of the optimum combinations of biocatalysts (provided from WP2), mediators (provided from WP3), electrode supports (provided from WP1) and immobilisation approaches (provided also from WP3). As a matter of fact, the progress within WP4 benefited from the concepts and delivered materials from WP1-3, with the initial aim to fulfil the specified technical criteria agreed achieved in deliverables D4.1-D4.5. Based on the fulfilment of D4.1-5 deliverables, in terms of current densities and operational onset potentials, the main focus of WP4 was directed towards reaching the power generation conditions required by the electronic design of the sensing device, and to secure its operation with satisfactory stability under physiological conditions in terms of substrate concentration, pH, halide concentration, as stipulated within WP5. The power generation conditions, required for further experiments within WP5, were set by the consortium to an operational cell potential of 600 mV and a minimum generated current of 40 µA. This had to be provided by fully integrated-operational BFCs combinations, for which the corresponding bioanodes and biocathodes relies on enzymatic concepts established by the consortium.
As a final testing of optimum combinations of bioanodes/biocathodes operating at pH 7 and in artificial serum medium, a join-experimental session was organised in April 2012 at RUB and included all the partners involved in the project. The final conclusive experiments (belonging to WP5) in Bochum demonstrated the feasibility of a self-powered device, operating under oxygen and glucose, and capable of sensing the concentrations of oxygen and glucose in an artificial serum, and further to wirelessly transfer the recorded signal to an external receiver.
WP5 - Fabrication, characterisation, and tests of microscale self-contained biodevices for biomedical application (Mah, NS, ULUND)
The WP5 contained 3 main tasks, which were fulfilled as described below.
Task 1 - Fabrication and optimisation of transmitting/transducer system with incorporated operational unit based on CC2430 microchip
At the beginning of the project some Partners (e.g. Mah, RUB, ULUND) were able to provide BFCs operating at 0.5 V or even less and providing few µW/cm2 power. These values were obtained from studies of not-optimised DET and MET based BFCs, which were fabricated based on available bioelements. The biodevices were characterised in simple buffer solutions and human physiological fluids, e.g. human serum. At the end of the project biodevices generating voltage of 0.6-0.8 V with few tens or even hundred microwatt/cm2 power densities were designed. The performance of biodevices was tested in simple buffer solutions and human physiological fluids, e.g. human blood, plasma, serum, and saliva.
One of the major tasks of NS was to address incompatible difference of voltage and power between the data transmitting systems and even optimised BFCs. First, an energy-harvesting module was designed. One of the tasks for the energy-harvesting module was to step up the low voltage the fuel cell delivers to a usable voltage for standard electronics. State of the art low power electronics (commercial available) still need quite a lot of energy, e.g. 90 000 µW for radio transmission and 2 000 - 20 000 µW for sampling. Thus, the energy-harvesting module also was designed to store energy and to control the activation of the standard electronics. This was necessary as standard electronics also draws too much power in stand-by mode. After the energy-harvesting module had stored enough energy to sample data and transmit one data package the radio module was activated from a control circuit via a switch. The CC2530 chip and measuring electronics was thus activated from the energy-harvesting module. The CC2530 has an integrated ADC, which was used to sample glucose and oxygen values from the measurement electronics. The data was directly transmitted to the receiver which was turned on all the time for receiving data. When the radio module had transmitted the information a signal was sent to the energy-harvesting module which then turned off the 'standard' electronics (radio and measurement electronics).
To integrate glucose sensing with the data transmission system a simple potentiostat was incorporated into the electronic device. One of the BFC electrodes serves as a counter and reference electrode. To minimise errors in glucose measurement, digital and analogue integration of the sensor signal were used and optimised. As was planned in the description of work the errors did not exceed 10% of the mean value from the bioanalyte signal.
Embedded software required to control the developed sensor system was developed. The main purpose of this software was to start up needed modules, sample sensor data and transmit the data safely to a receiving unit. This sequence of tasks should be as fast as possible to save power. Total duration achieved was 4.6 ms, whereof 3 ms was with radio transmitter activated. It should be noted that when the radio unit had completed the given task, a control signal was sent to the energy harvest module to shut down the switch providing power to the measurement electronics and radio transmitter. Thus the charging for next sample could commence immediately after finalising transmission. The transmitted package via the transmitter in the sensor was sent unsynchronised to the receiver to preserve energy and keep the sensor uptime low. Thus software for receiver had to operate continuously and wait for the next transmitted package from the receiver. However, this did not present a difficulty as the receiver connected to a computer did not consume more than 40 mA. When a package was received by the receiving radio the package was sent to an USB module, which was connected to the computer.
Task 2 - Fabrication of wireless devices for glucose or oxygen monitoring
Both glucose and oxygen sensitive wireless self-powered devices were fabricated as described in deliverables 5.5 and 5.6. Specifically, all different parts of a wireless self-powered biodevice, which were developed by Partners during the project, i.e. the electronic unit (containing energy harvesting module, potentiostat for a glucose or oxygen biosensor, and wireless communicating device, i.e. radio module), glucose or oxygen sensitive biosensor, and a sugar/oxygen BFC, were combined together into a functional unit. The general idea for electronics was to have the radio module turned off and when enough energy had been harvested by the energy harvesting module then wake up the radio module and the task at hand would then be: 1) to measure and sample data, 2) to establish a paired connection to a receiving radio base, 3) to send the measurement data via the radio, and finally, when the message has been transmitted, 4) to signal to the energy harvesting module to turn off the power supply and begin to store energy again. The receiver is then connected to a computer and is actively listening to new messages from the wireless sensor. When a message is received the data is extracted, stored, and displayed together with the time stamp for the data. These methods were adopted, implemented, and tested. For preliminary studies of developed demonstrators for glucose and oxygen biosensors, a single cell battery was used instead of BFCs. As glucose or oxygen sensitive biosensors AuNPs modified gold microwires (100 micrometre) with immobilised CtCDH or MvBOx were used. The performed preliminary tests revealed some limitations of the sensor electronics in the case of glucose sensing. Indeed, a new design for a glucose sensitive biodevice was developed. The old sensor electronics was primary developed for oxygen measurement, but with glucose the reference electrode is instead the anode. Thus, the negative power supply converter was not needed and the sensor electronics was reduced to a simple potentiostat meter with single power supply.Thus both prototypes to measure glucose and oxygen were fabricated and delivered to the consortium.
Task 3 -Device testing
In the frame of this task separate units of self-contained biodevices, i.e. electronic units, biocathodes, bioanodes, and complete BFCs, as well as two self-powered wireless biodevices were investigated including biodevice tests in simple buffer solutions and complex biological fluids, such as blood, plasma, serum, and saliva. Prior to these tests, the particular concentrations of two bioanalytes, viz. glucose and oxygen, in biological matrices was accurately measured using commonly used techniques, to enable correlation studies between the different devices to be performed.
Firstly, designed glucose oxidising and oxygen reducing biomodified electrodes were tested under cell culture conditions to investigate biocompatibility of the developed bioelectrocatalytic systems. For this purpose, oxygen reducing and glucose oxidising biodevices were designed in cell culture plates containing a permeable membrane as a bottom. The system was created by using disposable electrode arrays, 8W2x1E (applied biophysics, USA) which were modified prior to investigation to be used as biodevices. Each array contained eight wells with two planar gold electrodes each. The construction and the performance of the biodevices were optimised for several days monitoring of both bioanalytes in cell culturing condition. It was shown that catalytically inactive elements of biodevices, e.g. non-biomodified nanostructured surfaces based on carbon nanotubes, do not affect attachment and growth of cells on the surfaces. However, the introduction of biocatalysts, e.g.GOx or BOx enzymes, made surfaces strongly toxic even when biodevices are not electrically connected. The effect of GOx can be easily explained by the generation of hydrogen peroxide at GOx modified surfaces since both substrates of the enzyme are present in the solution, glucose and oxygen. In case of BOD the result cannot be explained by BOx catalytic reaction since in the absence of electron donors (without applied potential) the enzyme does not function. Further investigations are needed to understand the cell toxicity of certain nanostructured surfaces.
Secondly, the most successful constructions from WP4 were investigated in detail in biological fluids, such as blood, plasma, serum, and saliva, and limitations in their performances were studied and summarised. This included the tests of separate glucose oxidising (glucose sensitive biosensors and bioanodes) and oxygen reducing (oxygen sensitive biosensors and biocathodes) 3D nanobiodevices, as well as complete glucose/oxygen BFCs. It was shown that in simple air saturated buffer solutions bioanodes usually limited the performances of BFCs. However, when biodevice were investigated in complex air saturated physiological fluids significant decrease in biocathode performances was observed. Indeed, in real implanted situations, when free O2 concentration is much lower than 0.25 mM, BFCs might be limited by oxygen diffusion to the electrode nanostructured surfaces of biodevices.
Finally, two prototypes for monitoring of glucose and oxygen concentrations were tested using a battery instead of BFCs. From the very beginning of these tests the procedure to calibrate both systems was developed as described in deliverables 5.5 and 5.6. After the calibration of systems, the biodevices were tested. Specifically, the subsequent addition of bioanalytes in physiologically relevant concentrations (5 mM glucose or 0.25 mM oxygen) to the electrochemical cell resulted in the wirelessly measured signal, which was three or more times higher compared to the background value. To conclude, developed wireless electronic and operational units showed very stable and reliable wirelessly monitored signals. The next step was to perform analogous experiments powering the system with a BFC developed by the Consortium. This was done during the final meeting of the Consortium in Bochum (RUB University), Germany. A joint measurement session (5 working days) was organised with several tasks including the final test of two wireless self-powered biodevices, viz. glucose and oxygen sensitive self-contained biodevices. Two compartment BFCs were built using several different (mediator-based and mediator-less) bioanodes and biocathodes because of two main reasons, viz. (i) in order to achieve both current and voltage required for electronics, viz. 44 microampere and 0.57 V and (ii) in order to use all developed bioelectrodes to equalise contribution of all project partners. The following maximal parameters for the combined BFC were obtained: open circuit voltage of 0.73 V, 58 microampere at 0.67 V operating voltage. To increase the power of the BFC, lactose and oxygen at high concentrations, e.g. 10 mM and 0.25 mM, were used as biofuel and biooxidant in the anodic and cathodic compartments of the biodevice. Since lactose and oxygen are required to produce electric power their concentrations could not be significantly decreased during the tests.
The procedure to calibrate systems was exploited and operating voltage in the system was monitored by the external voltmeter. Variation of bioanalytes concentrations resulted in corresponding variation of wireless signals. Moreover, wireless signals from sugar and oxygen sensitive biodevices powered by BFC have been measured successfully for a long time, 1-2 hours. During this time several tens of packages of information were received wirelessly by the receiver, which was located 4 meters away from tested biodevices.
Potential impact:
The multidisciplinary 3D-NANOBIODEVICE consortium addressed several highly challenging, multi-industrial, and interdisciplinary objectives. To solve these,3D-NANOBIODEVICE, brought together leading scientists in the nanobiotechnology field specialising in investigations on enzyme-nanostructure interactions (e.g. enzyme-nanoparticle), integration of nanostructures and enzymes into 3D catalytic and electrically conducting structures for BFC applications and diagnostics. Nanostructures and biocatalysts (redox enzymes), knowledge on their interactions, design of BFCs using nanobiocomposites, and development of modern low voltage miniature electronics were some of the many outcomes of this consortium, outcomes that could not be achieved at any national level. Only European cooperation provided an opportunity to develop 3D nanobiostructure-based bio-electro-catalytic elements, which have a quality required to produce competitive self-contained wireless devices and evaluate these in biomedical application. One of our aims was to put the major fundamental goals of the project a better understanding of the interactions between bioelements and 3D nanostructures and fundamental principles for exploiting and developing electro-conducting nanoarchitectures to assemble highly efficient 3D bioelectrocatalytic structuresinto a practical test by assembling a device consisting of BFC, biosensor, and wireless radio transmitter and evaluating its performance in biological fluids. This was a practical step beyond the state of the art.
A convergence across industries and interdisciplinary studies resulted in breakthrough applications at the interfaces of energy and healthcare areas. The consortium contained two key industrial partners, viz. novozymes A/S (NZ) and Novosense AB (NS), both of which are eager to pick up the technological progress of the project for future commercialisation.
Novozymes A/S, a world leader in industrial biocatalysts production, is part of NovoNordisk, one of the leading producers of insulin and insulin pumps worldwide. The involvement of Novozymes in this project indicates that the company as a whole (i.e. NovoNordisk) is extremely interested, e.g. in acquiring 3D-nanobiodevicetechnology for the development of non-invasive glucose monitoring and wireless actuation of insulin pumps and their commercialisation in the health sector. Additionally, the development of 3D-nanobiobased BFCs is expected to contribute to a breakthrough in the energy area, where novozymes A/S has identified considerable potential to expand their commercial interests as an enzyme producer.
The second industrial partner of the consortium, novosense AB, is a young biomedical engineering company, with currently limited commercialisation potential simply because it is small. However, the company is working with wireless medical devices, which was a key issue of 3D-NANOBIODEVICE project. The company is developing wireless communication protocols to 'wire' medical instrumentation to databases at hospitals. This project enabled the company to develop means of minimising energy consumption for wireless transmission, to design an electric energy harvesting block and voltage amplifier, and thus progress the future integration of BFCs and biosensors into new wireless health care facilities. Additionally, the development of methods of integrating wireless medical devices to databases at hospitals has tremendously shortened the time needed to bring self-contained biodevices to the consumer market.
The potential commercial impact of the project is described in the preceding two paragraphs, which comes through direct collaboration with the industrial partners of the project, NS and NZ. However, the impact directions addressed in collaboration with the partners are much more general. More and more healthcare services and operations are conducted at homes and this trend converts healthcare system into a distributed biomedical industry, where biomedical devices are operated by patients or healthy humans. Healthcare innovations are accentuated by terms such as home based healthcare, mobile healthcare, point of care devices, wireless sensor networks, non-invasive monitoring, wearable and attachable biomedical devices, etc. The expected changes of healthcare systems to some extent can be compared with the changes of financial sector. We use credit cards and decide on credit limits, pay bills over the internet, and take loans without going to the bank. We enjoy financial autonomy. Surrounded by biomedical devices we will decide over our healthcare needs in the future much more than we do today. It is well realised that one of the bottlenecks to the use of distributed biomedical devices is a stable and reliable energy supply. It is hard to accept that we will change batteries in a number of biomedical devices at home every day or even every month. In this project we have been pursuing intensive research to develop and test BFCs as a reliable alternative for powering biomedical devices. We believe that BFCs will become an important power source in new generations of self-contained biomedical devices with self-powering and wireless capabilities. Thus, the progress achieved in this project on the development and combination of BFC with other biomedical devices have a very high impact on innovations promoting the establishment of distributed biomedical industry.
The ultimate goal of the consortium was to create self-contained miniature biomedical devices possessing wireless communication capabilities for implantable exploitations. Taking into account the world market for biosensors (ca. USD 15 billion in 2010 with an annual growth rate of 5%), it is very hard to overestimate the economic effect of this project related on modern biosensor technologies. Over half of the biosensors produced worldwide are employed in glucose meters. The possible economic effect of the project in terms of projected economic feasibility is based on glucose sensitive devices, both in short-term perspectives (i.e. in 3 years after the end of the project; in the year 2015) and in the long run (i.e. in 10 years after the end of the project, e.g. in the year 2022). Moreover, the total economic effect of the 3D-NANOBIODEVICE project is even higher because many other devices with possible commercial futures, e.g. different types of potentially implantable BFCs (mediator based and mediator less), electronic transmitter/transducer systems, oxygen sensitive microbiosensors, was also developed by the consortium. In general, 3D-nanobiodevicebioelectronic technologies, e.g. preparation of efficient 3D nanobiocatalytic structures or separate units, such as BFCs or biosensors, may provide valuable impact for the energy sector (BFCs), the environment (waste water treatment), and the development of in vitro diagnostics, whereas designing of microelectronics and modern software might accelerate the development of the Information technology (IT) sector.
According to the 'World diabetes market' report, diabetes affects approximately 200 million people worldwide and is expected to increase to 300 million diabetics by 2025. In many developed countries (e.g. USA, European Union) more than 5% of the population suffer from diabetes. It is the 6th most common cause of death as recorded on United States death certificates. With a diabetes epidemic underway, there exists strong growth opportunities for diabetes management tools, such as glucose meters. Over the years, glucose-monitoring meters have undergone a sea change, with recent entries featuring wireless and sensor technologies. Sales in United States, the largest market for glucose biosensors, are expected to reach USD 1.3 billion by the end of 2012. The data are based on the prediction that less-complicated devices produced by the consortium, i.e. microbiosensors, will pass clinical trials, be commercialised and account for less than 1 of the total market existing for glucose sensitive biosensors in 2015. Only in 2022 one can expect real commercialisation of wireless implantable self-powered biodevices for continuous glucose monitoring.
It is very hard to estimate the long-term perspectives for devices due to the existence of many different and unpredictable factors affecting their sales. However, taking into account the 5% per annum growth rate on the biosensor market as well as a robust management strategy, one can expect 2.5 times increased sales for microbiosensors in 2022 along with appearance of self-contained implantable biodevices for glucose monitoring on the European market, which will replace simple microbiosensors.
A long-term objective of the Project is to facilitate development of environmentally and body-friendly nanotechnologies for biomedical applications. Profound fundamental knowledge gathered during the project will provide the necessary knowledge for medical exploitation of 3D nanobiostructures as a basis for self-contained wireless biodevices consisting of a BFC, (bio)sensor(s), and a miniature signal transmitter system. The benefits to the society gained from the project can be foreseen as an acceleration of exploitation of knowledge-based products for sustainable innovations of the biomedical industry sector.The project facilitated the integration of biosensors, chips, and BFCs into integrated biomedical devices. It can be predicted that the developed technology will be used to improve quality of life and increase safety in case of widely occurring chronic diseases, when miniaturisation of self-contained and potentially implantable 3D nanobiostructure based devices is done. In the long-run, 3D nanobiostructure-based elements will be essential for constructing devices to be used for neuron/nerve stimulations and compensation of human disabilities.
Additionally, touching on the environmentally and body-friendly development of power sources in form of BFCs, the following can be emphasised. A great deal of the project has been focussed on enhancing BFC performance based on DET reactions between the enzymes and nanostructures. The realisation of DET principle allows more facile construction of BFC and also enables us to avoid additional active component of the system, e.g. mediators, which sometimes are debated as interfering with different redox cycles in living cells. The project has shown that DET based BFC can be as powerful as mediated systems and by this encourages future innovative developments of environmentally friendly DET based devices. This is a very important and fundamental impact arising from the results of this project.
All scientists participating in this project were exposed to a broad spectrum of both fundamental and technological knowledge and, equally important, they were trained in human resource management, gender and ethics related problems, importance of public acceptance of advanced science/technology, and a business/IP-minded thinking. As the final result, in the second part of the project (month 18-36) a close dialogue with two companies, which are established in the relevant sectors (Energy and biotechnology sectors), was organised by partner Mah with the support from MINC (www.minc.se) and Medeon (www.medeon.se).
The fundamental and technological knowledge developed during the project was discussed with CAVAC AB (www.cavac.se). Specifically, this young and ambitious Swedish company is interested in designing efficient and stable BFCs for different applications. Partner Mah was contacted by CAVAC as a possible party, which might provide technology to design efficient and stable cathodes. However, due to the lack of patents in this particular area (the Project was focused on biodevices instead of devices based on inorganic or organic catalysts) and financial constraints of the SME, no substantial agreement has been achieved so far.
Another example of commercialisation efforts from 3D-NANOBIODEVICE are in negotiations between the Finish company, VTT Biotechnology (www.vtt.fi) and Mah concerning a possible transfer of technology developed by researchers at Malmoe University during the project, specifically flexible, stable and efficient BFCs for extra vivo applications. Negotiations are ongoing and based on the patent WO/2011/117357 entitled: 'Flexible BFC, device and method'obtained by Mah with the financial support from 3D-NANOBIODEVICE.
Communication with the scientific community and the public at large was also an important task of 3D-NANOBIODEVICE. Accumulation of knowledge and its faithful dissemination to the society fully supports the ECs policy on integrated, safe, and responsible nanotechnology. The consortium hopes that these activities during the project enabled to increase both confidence and trust in the EC as a truly transparent and trustworthy communicator.
In addition to classical dissemination roots (publications in peer reviewed journals, conferences, workshops, symposiums, congresses, and public presentations) and novel/electronic means (web), results from the project were used in lectures, education programs, and other communication materials (newsletters, flyers, posters) via mass communication media. Detailed results on publications, patents, and disseminative activities of the consortium are presented in the final report, as well as summarised in Table 6.
List of websites: www.mah.se/3dnanobiodevice.
Self-contained wireless bioelectronic devices have huge scientific and practical importance for basic science and for possible applications in medicine, high-tech industry, military, and biocomputing. The anticipation of major breakthroughs in such a range of areas has resulted in significant research focus in such devices together with a rapid growth in the number of publications and patent applications in bioelectronics. The integration of biomaterials with electronic elements, such as electrodes, chips, field-effect transistors, and piezoelectric crystals, yields hybrid bioelectronic systems that may function as Biofuel cells (BFCs), biosensors, bioelectronic circuitry, and combinations thereof. The ultimate technological goal of our project 3D-NANOBIODEVICE was to construct a hybrid bioelectronic system, viz. potentially implantable self-contained biodevices working in biomatrices of different compositions, e.g. blood, serum, plasma, saliva, extracellular fluids, and cell cultures.
This goal was successfully accomplished. Specifically, at the very end of the project two proof-of-principle wireless self-powered biodevices were fabricated, calibrated, and tested. These devices contain glucose or oxygen sensitive biosensors connected to a transmitter/operating electronic device powered by BFCs. Signals from these devices, corresponding to varying concentrations of bioanalytes, are transferred to a back-end system, e.g. a computer with an electronic transducer for storage, processing, and integration of the biological information collected by the biosensors.
The main scientific goal of the project was to enhance understanding of the fundamental principles regarding the control of electron transfer reactions between nanoparticles, nanotubes, nanofibres, Os and Ru redox complexes, as well as their assemblies confined in ordered three-dimensional (3D) microscale networks combined with different bioelements, such as glucose oxidising and oxygen reducing enzymes, in order to solve one of the main obstacles of bioelectronics, i.e. the lack of (or poor) electronic communication between the biocomponents and the electronic elements. A number of novel 3D nanobiodevices were designed and tested, such as glucose and oxygen sensitive microbiosensors, nanostructured biocathodes and bioanodes, and their combinations with electronics (operating unit containing transmitter, potentiostat, electric power storage device and transducer) into autonomous and self-contained, potentially implantable devices powered by 3D BFCs. Theoretical modelling of the response of the sensors combined with a detailed experimental characterisation of the properties of the sensors were performed in glucose containing biological matrices of different composition and pH, e.g. blood, plasma, serum, and saliva. The required long-term, operational stability of the 3D nanostructured biodevices was achieved by choosing the appropriate bioelements from already available and from novel wild-type and specifically engineered highly active, stable, and halide resistant redox enzymes, such as glucose oxidase (GOx) and Cellobiose dehydrogenase (CDH), as well as high redox potential Blue multicopper oxidases (BMCO).
The self-contained biodevices that we developed are still macroscale in size. However, their miniaturisation is possible and such miniaturised biodevices can be used for continuous glucose and oxygen monitoring in different parts of the human body, thus improving the quality of life and increasing patient safety for such chronic conditions as diabetes and methemoglobinemia. Moreover, in the long-term, wireless self-powered biodevices based on nanobiostructures might be used for neuron/nerve stimulation to compensate or alleviate the effect of human disabilities as well as to treat chronic pain, cerebral palsy, and other conditions.
Beside the scientific and technological work, significant disseminative activities were carried out by the consortium. In addition to classical dissemination roots (publications in journals, conferences, symposiums, and congresses) and novel/electronic means (web), results from 3D-NANOBIODEVICE were used in lectures, education programs (summer schools, workshops, and colloquiums), and other communication materials (newsletters, flyers, posters) transmitted via mass communication media.
Project context and objectives:
The'Three-dimensional nanobiostructure-based self-contained devices for biomedical application' (3D-NANOBIODEVICE) project has been focused on several scientific, technological, and social objectives simultaneously.
One of the main scientific objectives was to enhance the understanding of the fundamental principles for controlling electron transfer reactions between gold nanoparticles (AuNPs), carbon nanotubes, carbon post materials (CPMs), conductive nano/microporous silicone and silicate (NMPSi and NMPSiT) chips, their assemblies confined into 3D microscale networks, and different bioelements, such as glucose oxidising (GOx and CDH) and oxygen reducing (laccase (Lc) and Bilirubin oxidase (BOx)) enzymes.
Another important scientific objective of the project was to understand experimental limits and fundamental differences in the performance of 3D nanobiostructures in different biomatrices, specifically human physiological fluids and cell based in vitro platforms versus simple buffer solutions.
The main technological objective of the project was to construct potentially implantable self-contained wireless biodevices for glucose and oxygen monitoring. Novel biodevices were constructed by combination of glucose and oxygen sensitive biosensors made from 3D nanobiostructured materials, and operated by an electronic unit containing a transmitter, operating system, potentiostat, and an electric energy harvesting device.
The last but not the least objective of the project, strongly oriented towards nanotechnology and biotechnology, which have usually topped most lists of areas where the 'next-big-thing' would come from, is an intensive and fair communication with the scientific community and the public at large, in full agreement with the European Commission (EC) strategic roadmap on communicating nanotechnology.
All scientific, technological, and disseminative objectives of the project were successfully accomplished as described in detail below. It is important to emphasise that the scientific and technological objectives of 3D-NANOBIODEVICElie somewhere between these two applications. By integrating fabricated nanobiostructure based biodevices and electronic elements into functional biomedical devices with a wireless transmission feature and testing the proof-of-concept for wireless glucose and oxygen sensing in real biological fluids, 3D-NANOBIODEVICE project stepped far beyond the state of the art.
Nanowiring of appropriate redox enzymes with AuNPs, carbon nanotubes, CMPs, proper surface modifications, and use of Os and Ru redox complexes, were chosen as a major direction to solve main obstacles in the area of bioelectronics, i.e. poor electronic communication between the biocomponents and the electronic elements along with insufficient operational stability. To produce 3D bioelectrodes with superior characteristics mathematical modelling of their performance was performed and the results obtained from calculations were compared against experimentally determined parameters. From the modelling and characterisation results, optimisation of the biodevices (glucose and oxygen sensitive biosensors, bioanodes, and biocathodes) was done to increase their efficiency and stability.
Such an ultimate technological goal, as the construction of potentially implantable self-powered wireless biodevices working in biomatrices of different compositions and pHs, could be achieved only in the very end of 3D-NANOBIODEVICE. The final outcome of the project has been broken down to smaller tasks, which can be divided into seven main groups:
i) development of electronic parts of the devices, their combination into the functional transmitter/receiver system, and development of the requisite software;
ii) fabrication of stable 3D-nanostructures;
iii) production of active bioelements;
iv) design of Os or Ru containing redox polymers;
v) combination of nanostructures and redox enzymes into efficient nanostructured biomodified electrodes, which could serve as glucose and oxygen sensitive biosensors, sugar oxidising bioanodes, and oxygen reducing biocathodes;
vi) fabrication and characterisation of direct ET (DET) and mediated ET (MET) based BFCs;
vii) design and testing of self-contained biodevices for glucose and oxygen monitoring.
Indeed, these scientific and technological steps were successfully reached by the consortium as detailed below:
1.1. development of a wireless transmission system with voltage multiplayer converting 0.57 V to 2.5 V integrated with capacitive power storage;
1.2. design of a micro-potentiostat integrated with glucose and oxygen biosensors and wireless transmission system;
1.3. creation of a data transmission system with optimised data transfer and antenna for data transfer over distances of more than 3 metres;
1.4. development of optimised hardware and software components as a basis for a self-contained measurement-transmitting device.
2.Electroconducting 3D nanoarchitectures were fabricated and characterised morphologically and electrochemically:
2.1. NMPSi and NMPSiT chips of area 6x6 mm2 and of depth 10-100 micrometres;
2.2. ordered assembles of nanoparticles (OAN) with NPs sizes varying from 2 to 200 nm;
2.3. hierarchical two-generation carbon nanotube-based materials (HTGCNT) 10-100 µm deep;
2.4. CPM arrays with 6x6 mm2 areas and 10-100 µm deep structures;
2.5. mesoporous gold electrodes.
3.1.Available glucose oxidising and oxygen reducing enzymes were isolated on preparative scale (up to 500 mg), purified to homogeneity, and characterised biochemically and kinetically:
3.1.1. anodicbioelements: both GOx and CDH, > 100 mg;
3.1.1. cathodicbioelements: BMCO, viz. Lcs and BOx, > 100 mg;
3.2. newly screened and designed bioelements were produced, purified, and delivered to the consortium:
3.2.1.anodicbioelements: both GOx and CDH, > 50 mg;
3.2.2. cathodicbioelements: chloride resistance high redox potential Lcs, > 50 mg.
4. Os or Ru containing redox polymers were produced, tested, and delivered to consortium. A large library of redox polymers was also designed.
5.3D biodevices functioning as glucose and oxygen sensitive biosensors, sugar oxidising bioanodes, and oxygen reducing biocathodes, were fabricated by biomodification of 3D nanostructures with suitable bioelements.
6.1.Unoptimised DET and MET based BFCs were fabricated based on available bioelements. The biodevices were characterised in simple buffer solutions and human physiological fluids, e.g. human serum.
6.2.Optimised DET and MET based BFCs were designed based on fabricated electroconducting 3D nanoarchitectures modified with novel engineered bioelements. The performance of biodevices was tested in simple buffer solutions and human physiological fluids, e.g. human blood, plasma, serum, and saliva.
7.Two wireless self-powered biodevices for glucose and oxygen monitoring were designed and tested.In addition to technological tasks the fundamental principles of the function of electroconducting biodevices based on 3D nanobiostructures were studied and some experimental limits in their performances were clarified as described below:
a) Efficient motionless 3D nanobiostuctures based biodevices for oxygen reduction perform in mass-transfer limited regime, i.e. with maximal current densities up to 0.5 mA cm-2, whereas most CDH modified bioelectrodes were limited by the activity of redox enzymes immobilised on the electrode surface. Contrary to CDH modified electrodes, some GOx modified 3D nanobiostructures also operate in mass-transfer limited regime, i.e. with maximal current densities up to 5 mA cm-2.
b) Most of the 3D nanobiosturctures based biodevices for glucose oxidation operated at quite high potentials (up to 0.5 V vs. NHE), which is not suitable for biosensing purposes and BFC applications. Several possibilities to decrease the overpotential for sugar oxidation of CDH- and GOx modified 3D electrodes were undertaken, viz. mutagenesis of redox enzymes and appropriate choice of redox mediators using a design library of Os and Ru containing redox polymers. Contrary to CDH- and GOx modified electrodes, motionless 3D nanobiostuctures based biodevices for oxygen reduction operated at potentials very close to redox equilibrium potentials of the oxygen/water couple at certain conditions (e.g. 0.7 V vs. NHE at physiological pH values).
Another scientific objective of 3D-NANOBIODEVICE entailed the investigation of fabricated and characterised 3D-nanobiostructures in human blood, plasma, saliva, and cell based in vitro platforms vs. simple buffer solutions. Specifically:
a) Needed equipment for investigation of human physiological fluids were installed, volunteers (donors) were identified, and samples were collected. This was done in accordance with ethical issues and biomedical protocols presented in detailed project description (annex I).
b) Investigations of designed nanobiostructures in blood, plasma, serum, and saliva were performed. It was shown that 3D bioelectrocatalytic structures for oxygen electroreductionlose efficiency and stability in complex physiological fluids because of the inhibition/deactivation of redox enzymes (Lc and BOx) by different organic and inorganic substances. In addition, electrochemical oxidation of different interfering compounds, primarily ascorbate occurred on the nanostructured electrodes. Two major strategies towards overcoming the expected limitations of biocathode performance can be proposed, viz. (i) blocking of the bare nanostructured surface to disable direct contact with interfering molecules and (ii) use of another electrode material, which is inert with regard to ascorbateelectrooxidation. The latter approach is quite difficult to realise since interfering molecules, e.g. ascorbate, can be efficiently electrooxidised at quite low overpotentials on most conductive electrodes. To block ascorbate molecules from bare surfaces, negatively charged polymers can be used. However, a significant efficiency decrease of the essentially membrane based biocathode compared to membrane-less biodevices is expected, owing to mass-transfer limitations. Thus, one of the best approaches is to block nanostructured surfaces with a dense monolayer of a biocatalyst using oriented enzyme immobilisation on modified electrode surfaces, as has been already done in the case of Lc-based biocathodes (partner CSIC).
In contrast, glucose oxidising 3D-nanobiostructures can, in principle, be activated by inorganic compounds, e.g. calcium ions, as was shown by Partner ULUND. However, the concentration of these compounds in human fluids is not sufficient to enable activation, while the low substrate content of biofluids (glucose, lactose, etc.) resulted in quite inefficient performance of CDH-based nanobiostructures. One of the possible solutions to improve efficiency of bioanodes in physiological biomatrices is the use of even more efficient biocatalysts, e.g. engineered CDH with low KM and high kcat values for glucose. Another approach is to increase the coulombic efficiency of anodic biodevices. The majority of currently developed biodevices extract only two electrons from glucose or lactose molecules, while much more is really possible. For instance, complete oxidation of glucose can give 24 electrons and thus approaching complete oxidation of glucose is important scientific challenge into improving the power output from bioanodes operating in energetically poor (with low amount of biofuels, e.g. glucose) physiological fluids, e.g. human saliva.
Additional testing of biomodified electrodes was performed in cell culture conditions to investigate their biocompatibility. For this purpose, oxygen reducing and glucose oxidising biodevice were designed in cell culture plates containing a permeable membrane at the bottom. The system was created by using disposable electrode arrays, 8W2x1E (applied biophysics, USA) which were modified prior to investigation biodevices. Each array contained eight wells with two planar gold electrodes each. The construction and the performance of the biodevices were optimised for several days by monitoring of both analytes under cell culture conditions. The catalytically inactive elements of biodevices, e.g. non-biomodified nanostructured surfaces based on carbon nanotubes, did not affect attachment and growth of cells on the surfaces. However, the introduction of biocatalysts, e.g. GOx or BOx enzymes rendered the surfaces strongly cytotoxic even when the biodevices were not electrically connected. The effect of GOx can be easily explained by the generation of hydrogen peroxide at GOx modified surfaces since both substrates of the enzyme are present in the solution, glucose and oxygen. In case of BOD the result cannot be explained by BOx catalytic reaction since in the absence of electron donors (without applied potential) the enzyme does not function. Further investigations are needed to understand the cell toxicity of the nanobiostructured surfaces.
It should be emphasised that wireless self-powered 3D nanobiostructure based devices developed in the frame of the project are still macroscale. Moreover, miniaturisation of electronics is also need. However, the results of the project including proof-of-principle tests show, without a doubt, that miniature self-contained biodevices can be used for continuous glucose and oxygen monitoring in different parts of the human body, thus improving quality of life and increasing patient safety in the case of widely prevalent chronic diseases such as diabetes and methemoglobinemia. Moreover, in the long term, 3D nanobiostructure based elements will be essential for constructing devices to be used for neuron/nerve stimulations and compensation of human disabilities.
Besides scientific and technological work strong communication activities were carried out by the Consortium. In addition to classical dissemination roots (publications in journals, conferences, and workshops) and novel/electronic means (web), results from '3D-NANOBIODEVICE' were used in lectures, education programs, and other communication materials (newsletters, flyers, posters) via mass communication media.
Project results:
All the work of the consortium has been done in strict observance with the structure of annex I of the grant agreement following the project objectives and description of work. The detailed plan of the work was described in five Workpackages (WPs).
WP1 - Fabrication and characterisation of microscale 3D electrodes (AU, RUB, UL, ULUND)
One of the main aims of this WP was to produce conductive, high surface area supports for the immobilisation of enzymes for use in biosensors and BFCs. High surface area supports are essential for the successful immobilisation of enzymes at loadings that can deliver the current and power requirements of the devices needed for self-contained wireless bioelectronic devices. The main objective of this WP was to produce conducting 3D nanostructures with controlled nanodimensions, surface chemistries and surface energies/curvatures for efficient performance of enzymes in 3D nanostructures. Four approaches were planned: (i) the fabrication of carbon post materials (CPM), (ii) the formation of conductive NMPSi and NMPSiT chips, (iii) the synthesis of hierarchical two-generation carbon nanotube materials (HTGCNT) grafted onto graphite electrodes and (iv) the fabrication of ordered assembles of nanoparticles (OAN) and nanoporous gold chips (NGC). Devices using each of these approaches have been prepared and characterised as described in the following sections.
WP2 - Engineering of bioelements by rational design and directed evolution (BOKU, CSIC, NZ, INBI)
The contribution of WP2 to the project involved the production of existing glucose oxidising (anode) and oxygen reducing (cathode) enzymes, as well as new wild-type enzymes obtained by screening, and the development of protein engineered variants with improved properties. The aim of this WP is to provide the consortium with the best bioelements to design efficient and stable BFCs, as well as sensitive and selective biosensors.
As a starting point, a collection of known enzymes was produced in purified form (at ~100 mg scale) and supplied to the consortium partners for initial testing. These included a variety of CDH from myriococcumthermophilum (MtCDH) and Humicolainsolens (HiCDH), and also the well-known GOx from aspergillusniger (AnGOx), which has been used in several commercial glucose biosensors. This set of enzymes has a wide range of properties (pH, glucose activity, thermal stability) and allowed for the construction and testing of bioanodes and glucose sensitive biosensors utilising both MET and DET systems, under different reaction conditions, as well as different immobilisation strategies. For testing the construction and initial performance of biocathodes, Lcs from myceliophthorathermophila (MtLc), streptomycescoelicolor (ScLc), trameteshirsuta (ThLc), rhusvernicifera (RvLc), as well as a BOx from myrotheciumverrucaria (MvBiOx), were produced, purified, and sampled. These enzymes also cover a range of characteristics, with high-, medium- and low-redox potential enzymes, of different pH optima and susceptibility to chloride ions (a common issue with laccase-based systems). Despite the variety observed in this set of available enzymes, there were a number of limitations of the bioelements that needed to be overcome to obtain an optimal bioelectrode working under physiological conditions (pH 7.4 glucose concentration of approximately 5 mM and chloride concentration of approximately150 mM). These limitations were addressed by screening for new wild-type enzymes, as well as optimising performance of the best available enzymes by protein engineering.
The sourcing of new wild type enzymes commenced using a Prussian blue-based screening assay for novel carbohydrate oxidoreductases with improved glucose turnover at pH 7.4. This process was successfully developed and validated by the subcontractor Prof. M. Rabinovich from the A.N. Bach Institute of Biochemistry, Moscow. In the following screening of several hundred fungal cultures 15 carbohydrate oxidoreductase producing fungal species were identified 7 of which were characterised and the results published (Harreither et al., 2011; Sygmund et al., 2011a). Of these enzymes, six were CDHs and one an FAD-dependent glucose dehydrogenase (GDH). After investigating the molecular and catalytic properties MtCDH, dichomerasaubinetii CDH (DsCDH), and corynascusthermophilus CDH (CtCDH) and GDH from glomerellacingulata (GcGDH) were produced on a large scale (approximately 300 mg) and distributed to the cooperating partners for testing in combination with existing electrodes. These CDHs have a significantly improved glucose turnover at pH 7.4 compared to the previously available basidiomycetous CDHs (Harreither et al., 2012).
Further improvements of DET-anode biocatalysts were pursued by means of genetic engineering using a rational, structure-based approach. MtCDH was successfully used as template to improve the Intramolecular electron transfer (IET) between the flavodehydrogenase domain and the cytochrome domain to achieve an overall faster electron transfer from the substrate to the electrode. The main steps in this process were the comparative modelling of the enzyme's structure, docking experiments to evaluate the domain interaction, the identification of factors increasing the IET, site-directed mutagenesis, recombinant production in the expression host pichiapastoris, purification by a multi-step chromatographic procedure, steady-state and transient-state kinetic measurements, and finally the evaluation of the obtained enzyme variants. Beneficial mutations were combined and final variants carried up to seven mutations which increased the IET up to 3.5-fold. In another subtask the glucose turnover in MtCDH and CtCDH was increased by mutating the active site and a combination of certain mutations. The catalytic efficiency of both CDHs for glucose turnover was increased by a factor of 5. Additionally, the catalytic efficiency for maltose, which is an interfering substance for glucose biosensors, could be reduced 100-fold.
Another subtask involved the production of a lower redox potential form of the cytochrome domain in CDH to increase the open circuit potential of the developed BFCs. For this purpose 13 positions in the primary and secondary amino acid shell around heme b cofactor of the cytochrome domain were targeted by site-directed mutagenesis. The rationale was to (i) introduce a covalent bond to the cofactor to change the b-type heme into a c-type heme, which has a lower redox potential, or (ii) reduce the number of electron withdrawing residues around the hemestetrapyrrole ring. The obtained results are ambivalent: although several enzyme variants show a lower redox potential (50 to 60 mV vs. NHE) than the wild-type enzyme (100 mV vs. NHE) the IET is also greatly reduced and limits the electron transfer rate. By using the obtained variants, the open circuit potential of the BFC could be increased by a maximum of 50 mV, but the current output would be 10 50-fold lower.
On a parallel track, protein engineering of HiCDH was employed to improve the activity on glucose at 5 mM concentration and pH 7. Initially, molecular modelling and docking experiments were performed to obtain a structural model of the enzyme and identify suitable positions for mutagenesis. The diversity was obtained by site-directed mutagenesis, targeted primary to residues in the vicinity of the active-site, and the resulting variants were screening using two assays: cytochrome c and DCPIP as electron acceptors, as measures of DET and MET, respectively. The best hits from the variant screening were expressed in Aspergillusoryzae, purified and characterised to obtain kinetic constants. The optimisation of HiCDH resulted in variants with up to 30-fold improved activity on glucose (5 mM), at pH 7, and catalytic rates above 2 s-1 (at 5 mM glucose).
Also, some optimisation work was done to improve the dehydrogenase activity of AnGOx, as a measure to improve its activity in MET electrodes. Structure-based mutagenesis with modifications on the active site resulted in improved variants with up to 3-fold higher activity on DCPIP and up to 90% reduced oxidase activity (an unwanted competing reaction). Finally, GDH from G. cingulata (GcGDH), which has a high glucose turnover (approximately 500 s-1) at pH 7.4 was employed in electrodes developed for mediated electron transfer (MET). To increase the availability of the enzyme it was cloned and recombinantly expressed in P. pastoris and kinetically characterised (Sygmund et al., 2011b). Additionally, special mutations were introduced to make the enzyme susceptible for covalent attachment to the electrode via linkers to increase electron transfer rates.
In order to obtain a superior enzyme for the second half-reaction (reduction of oxygen), taking place at the cathode, engineering of two high-redox potential Lcs from basidiomycetes PM1 and pycnoporuscinnabarinus (PM1Lc and PcLc, respectively) was undertaken. The initial strategy comprised the replacement of the native signal peptides by the alpha factor preproleader and the joint evolution of the fusion genes to give rise to detectable secretion levels in S. cerevisiae.
The PM1Lc was subjected to 8 rounds of evolution and screening, engineering a protein mutant (OB-1) with a total laccase activity improvement of 34,000-fold over the parent type (8 mg/L of secreted protein) (Figure 23). The OB-1 mutant harbored mutations V162A, H208Y, S224G, A239P, D281E, S426N and A461T in the mature protein whereas V(10)D, N(23)K and A(87)T occurred at the preproleader. OB-1 was fairly active and stable in terms of temperature (T50 = 73 degrees of Celsius), pH profile and the presence of co-solvents. The PcLc evolution was carried out in the presence of natural and artificial redox mediators enhancing the secretion levels 40-fold along with a 14-fold increase in kcat. The evolved preproleaders were useful for the secretion of native laccases in yeast and constituted a valuable tool for further protein engineering. The evolved PcLc was also highly secreted by other heterologous host (Aspergillusniger) at levels as high as 23 mg/L. The evolved PcLcharbored mutations N208S, R280H, N331D, D341N and P394H in mature protein whereas showed A)(9)D, F(48)S, S(58)G, G(62)R and E(86)G at the preproleader. PM1Lc and PcLc evolved laccases were subjected to a chimeragenesis protocol to shuffle the properties of both enzymes finding new variants with novel pH profiles and/or improved thermal stabilities.
The second challenge of evolving these laccases was to improve the activity at neutral pH and in the presence of high concentrations of chloride ions (human blood resistant). The PM1Lc evolved for functional expression and activity (OB-1 mutant) was selected as starting point to tailor a human blood tolerant laccase. A screening protocol based on the use of surrogate blood (blood buffer with similar biochemical composition as blood but in the absence of cells and coagulation agents) was employed to stress enzymes in the presence of chlorides at alkaline pH.To make a breakdown of the evolved properties several re-screens were incorporated in each generation finding out the individual improvements in terms of inhibition (vs. halides and hydroxides) as well as stability. After four cycles of evolution and semi-rational approaches (including several site- and saturation- mutagenesis experiments in different positions of the OB-1 gene) an ultimate variant (ChU-B mutant) was engineered containing 2 mutations located at the second coordination sphere of the T1 Cu site (F396I and F454G). The evolved mutant was biochemically characterised from two glycoforms with similar features and slight differences in glycosylations degrees (with molecular weights of 60310 and 58052 Da, respectively). Regardless of the isoform, the pH profile was noticeably shifted as main consequence of the directed evolution (even changing the maximum pH for activity vs. phenolic compounds form 4.0 to 5.0). The residual activity of the ChU-B mutant at pH 7.4 (pH of blood) was 7% for ABTS and 23% for DMP. We are not aware about reports of other HRPLs showing activity at pH above 7.0. ChU-B mutant enhanced its I50 for chlorides from 169 mM up to 1024 mM. A less pronounced effect was detected for fluorides (which were not targeted for evolution) with a change in the I50 from 69 to 109 mM, for parent and ChU-B mutant respectively. In general terms, the ChU-B mutant showed turnovers in blood buffer of 427 and 143 mol product mol laccase-1 min-1, for DMP and ABTS, respectively. Neither the parent nor other HRPLs tested in our laboratory (e.g. TrameteshirsutaLc) display activity under such conditions. In collaboration with partner 7B, the mutant was preliminarily characterised in terms of electrochemical responses and direct electron transfer has been detected at electrodes in blood buffer. Finally, in collaboration with Mah, the mutant was tested in real human blood and plasma, showing activity (measured as consumption of oxygen and using ascorbic acid as reducing substrate). This last mutant was over-expressed in pichiapastoris in collaboration with BOKU and supplied to the consortium for different trials (around 25 mg of pure mutant protein was delivered to the consortium in the final meeting). The mutant was successfully tested in the prototype designed during the final workshop, which took place at Bochum, Germany (RUB).
In summary, the results from WP2 provided improved glucose-oxidising enzymes both for DET (engineered CDHs) and MET (GcGDH and engineered AnGOx), as well as oxygen-reducing enzymes, viz. laccase variants with high redox potential and activity in blood buffer, for the construction of glucose and oxygen sensitive biosensors, as well as glucose/oxygen BFCs, much superior to the available bioelements at the start of the project.
WP3 -Biomodification of the selected 3D electrodes (Mah, AU, RUB, UL, CSIC, ULUND, NUIG, US)
The main objective of WP3 is to investigate, and report on, methods for electronically addressing glucose oxidising and oxygen reducing enzymes (from WP2) in 3D structures (from WP1). The programme was split into two tasks, the first focused on synthesis and characterisation of a range of redox complexes and polymers that can mediate electron transfer between enzymes and electrodes, and the second task focused on screening approaches for modification of 3D electrode surfaces with components (enzyme, and mediator if necessary) to provide preliminary results on enzyme electrode signal, and signal stability, as a precursor to further tests on biosensor and biopower device operation in WP4.
(i) Generation and characterisation of redox polymers (RUB and NUIG)
Redox complexes, and redox polymers, are used as components of enzyme electrodes in biosensor and biopower devices to enable efficient mediated electron transfer (MET) between enzyme and electrode (electronic wiring of enzyme). A robotic synthesiser system, and/or combinatorial parallel synthesis, using a range of monomeric components and polypyridyl ligands provided for polymer and redox complex libraries. Combination, through parallel synthesis, of elements from the two libraries were then used to prepare a range of redox polymers with a diversity of redox potential, and physicochemical properties, that could be tailored for the application desired, such as redox enzyme active site potential or immobilisation approach. Preliminary characterisation of the polymers, the redox complexes and the redox polymers enabled reporting of the polymer composition (Table 1) and redox complex/polymer potential (Os2+/3+ transition).
Initial testing of enzyme electrodes prepared using co-immobilisation of redox polymers with model enzymes focused on simple physisorption onto solid (carbon) electrodes and evaluation of the bioelectrocatalytic currents for substrate oxidation (for sugar-oxidising enzymes) or reduction (for oxygenase enzymes). For example RUB have characterised the response for bioelectrocatalytic reduction of oxygen in thin films of selected redox polymers co-immobilised with the Trametes hirsute laccase as a function of enzyme loading.
NUIG investigated glucose oxidation by thin films of redox polymer co-immobilised on graphite with glucose oxidase in different ratios, using different crosslinking agents in the presence and absence of added carbon nanotubes.
(ii) Screening for various procedures for modification/activation of electrode surfaces for facilitating immobilisation/ET with biocomponents and/or mediators
Four types of 3D-nanostructures were fabricated in WP1: (i) CMP, (ii) NMPSi and NMPSiT, (iii) HTGCNT, and (iv) OAN. Work on NMPSi and NMPSiT was not pursued as these electrodes were not sufficiently stable (silicate materials) or did not provide the performance needed (silicon electrodes). These materials were replaced with porous gold modified electrodes which were shown to provide high surface areas (WP1).
Finally, as part of WP3 flexible surface attachment chemistry (US) for carbon surfaces was developed using a high-yielding maleimide coupling of enzyme to pre-treated carbon surfaces, as reported on in deliverable 3.3. WP4 - Characterisation and optimisation of microscale 3-D biodevices (AU, RUB, UL, CSIC, ULUND, NUIG, US)
The aim of WP4 represented the evaluation of the optimum combinations of biocatalysts (provided from WP2), mediators (provided from WP3), electrode supports (provided from WP1) and immobilisation approaches (provided also from WP3). As a matter of fact, the progress within WP4 benefited from the concepts and delivered materials from WP1-3, with the initial aim to fulfil the specified technical criteria agreed achieved in deliverables D4.1-D4.5. Based on the fulfilment of D4.1-5 deliverables, in terms of current densities and operational onset potentials, the main focus of WP4 was directed towards reaching the power generation conditions required by the electronic design of the sensing device, and to secure its operation with satisfactory stability under physiological conditions in terms of substrate concentration, pH, halide concentration, as stipulated within WP5. The power generation conditions, required for further experiments within WP5, were set by the consortium to an operational cell potential of 600 mV and a minimum generated current of 40 µA. This had to be provided by fully integrated-operational BFCs combinations, for which the corresponding bioanodes and biocathodes relies on enzymatic concepts established by the consortium.
As a final testing of optimum combinations of bioanodes/biocathodes operating at pH 7 and in artificial serum medium, a join-experimental session was organised in April 2012 at RUB and included all the partners involved in the project. The final conclusive experiments (belonging to WP5) in Bochum demonstrated the feasibility of a self-powered device, operating under oxygen and glucose, and capable of sensing the concentrations of oxygen and glucose in an artificial serum, and further to wirelessly transfer the recorded signal to an external receiver.
WP5 - Fabrication, characterisation, and tests of microscale self-contained biodevices for biomedical application (Mah, NS, ULUND)
The WP5 contained 3 main tasks, which were fulfilled as described below.
Task 1 - Fabrication and optimisation of transmitting/transducer system with incorporated operational unit based on CC2430 microchip
At the beginning of the project some Partners (e.g. Mah, RUB, ULUND) were able to provide BFCs operating at 0.5 V or even less and providing few µW/cm2 power. These values were obtained from studies of not-optimised DET and MET based BFCs, which were fabricated based on available bioelements. The biodevices were characterised in simple buffer solutions and human physiological fluids, e.g. human serum. At the end of the project biodevices generating voltage of 0.6-0.8 V with few tens or even hundred microwatt/cm2 power densities were designed. The performance of biodevices was tested in simple buffer solutions and human physiological fluids, e.g. human blood, plasma, serum, and saliva.
One of the major tasks of NS was to address incompatible difference of voltage and power between the data transmitting systems and even optimised BFCs. First, an energy-harvesting module was designed. One of the tasks for the energy-harvesting module was to step up the low voltage the fuel cell delivers to a usable voltage for standard electronics. State of the art low power electronics (commercial available) still need quite a lot of energy, e.g. 90 000 µW for radio transmission and 2 000 - 20 000 µW for sampling. Thus, the energy-harvesting module also was designed to store energy and to control the activation of the standard electronics. This was necessary as standard electronics also draws too much power in stand-by mode. After the energy-harvesting module had stored enough energy to sample data and transmit one data package the radio module was activated from a control circuit via a switch. The CC2530 chip and measuring electronics was thus activated from the energy-harvesting module. The CC2530 has an integrated ADC, which was used to sample glucose and oxygen values from the measurement electronics. The data was directly transmitted to the receiver which was turned on all the time for receiving data. When the radio module had transmitted the information a signal was sent to the energy-harvesting module which then turned off the 'standard' electronics (radio and measurement electronics).
To integrate glucose sensing with the data transmission system a simple potentiostat was incorporated into the electronic device. One of the BFC electrodes serves as a counter and reference electrode. To minimise errors in glucose measurement, digital and analogue integration of the sensor signal were used and optimised. As was planned in the description of work the errors did not exceed 10% of the mean value from the bioanalyte signal.
Embedded software required to control the developed sensor system was developed. The main purpose of this software was to start up needed modules, sample sensor data and transmit the data safely to a receiving unit. This sequence of tasks should be as fast as possible to save power. Total duration achieved was 4.6 ms, whereof 3 ms was with radio transmitter activated. It should be noted that when the radio unit had completed the given task, a control signal was sent to the energy harvest module to shut down the switch providing power to the measurement electronics and radio transmitter. Thus the charging for next sample could commence immediately after finalising transmission. The transmitted package via the transmitter in the sensor was sent unsynchronised to the receiver to preserve energy and keep the sensor uptime low. Thus software for receiver had to operate continuously and wait for the next transmitted package from the receiver. However, this did not present a difficulty as the receiver connected to a computer did not consume more than 40 mA. When a package was received by the receiving radio the package was sent to an USB module, which was connected to the computer.
Task 2 - Fabrication of wireless devices for glucose or oxygen monitoring
Both glucose and oxygen sensitive wireless self-powered devices were fabricated as described in deliverables 5.5 and 5.6. Specifically, all different parts of a wireless self-powered biodevice, which were developed by Partners during the project, i.e. the electronic unit (containing energy harvesting module, potentiostat for a glucose or oxygen biosensor, and wireless communicating device, i.e. radio module), glucose or oxygen sensitive biosensor, and a sugar/oxygen BFC, were combined together into a functional unit. The general idea for electronics was to have the radio module turned off and when enough energy had been harvested by the energy harvesting module then wake up the radio module and the task at hand would then be: 1) to measure and sample data, 2) to establish a paired connection to a receiving radio base, 3) to send the measurement data via the radio, and finally, when the message has been transmitted, 4) to signal to the energy harvesting module to turn off the power supply and begin to store energy again. The receiver is then connected to a computer and is actively listening to new messages from the wireless sensor. When a message is received the data is extracted, stored, and displayed together with the time stamp for the data. These methods were adopted, implemented, and tested. For preliminary studies of developed demonstrators for glucose and oxygen biosensors, a single cell battery was used instead of BFCs. As glucose or oxygen sensitive biosensors AuNPs modified gold microwires (100 micrometre) with immobilised CtCDH or MvBOx were used. The performed preliminary tests revealed some limitations of the sensor electronics in the case of glucose sensing. Indeed, a new design for a glucose sensitive biodevice was developed. The old sensor electronics was primary developed for oxygen measurement, but with glucose the reference electrode is instead the anode. Thus, the negative power supply converter was not needed and the sensor electronics was reduced to a simple potentiostat meter with single power supply.Thus both prototypes to measure glucose and oxygen were fabricated and delivered to the consortium.
Task 3 -Device testing
In the frame of this task separate units of self-contained biodevices, i.e. electronic units, biocathodes, bioanodes, and complete BFCs, as well as two self-powered wireless biodevices were investigated including biodevice tests in simple buffer solutions and complex biological fluids, such as blood, plasma, serum, and saliva. Prior to these tests, the particular concentrations of two bioanalytes, viz. glucose and oxygen, in biological matrices was accurately measured using commonly used techniques, to enable correlation studies between the different devices to be performed.
Firstly, designed glucose oxidising and oxygen reducing biomodified electrodes were tested under cell culture conditions to investigate biocompatibility of the developed bioelectrocatalytic systems. For this purpose, oxygen reducing and glucose oxidising biodevices were designed in cell culture plates containing a permeable membrane as a bottom. The system was created by using disposable electrode arrays, 8W2x1E (applied biophysics, USA) which were modified prior to investigation to be used as biodevices. Each array contained eight wells with two planar gold electrodes each. The construction and the performance of the biodevices were optimised for several days monitoring of both bioanalytes in cell culturing condition. It was shown that catalytically inactive elements of biodevices, e.g. non-biomodified nanostructured surfaces based on carbon nanotubes, do not affect attachment and growth of cells on the surfaces. However, the introduction of biocatalysts, e.g.GOx or BOx enzymes, made surfaces strongly toxic even when biodevices are not electrically connected. The effect of GOx can be easily explained by the generation of hydrogen peroxide at GOx modified surfaces since both substrates of the enzyme are present in the solution, glucose and oxygen. In case of BOD the result cannot be explained by BOx catalytic reaction since in the absence of electron donors (without applied potential) the enzyme does not function. Further investigations are needed to understand the cell toxicity of certain nanostructured surfaces.
Secondly, the most successful constructions from WP4 were investigated in detail in biological fluids, such as blood, plasma, serum, and saliva, and limitations in their performances were studied and summarised. This included the tests of separate glucose oxidising (glucose sensitive biosensors and bioanodes) and oxygen reducing (oxygen sensitive biosensors and biocathodes) 3D nanobiodevices, as well as complete glucose/oxygen BFCs. It was shown that in simple air saturated buffer solutions bioanodes usually limited the performances of BFCs. However, when biodevice were investigated in complex air saturated physiological fluids significant decrease in biocathode performances was observed. Indeed, in real implanted situations, when free O2 concentration is much lower than 0.25 mM, BFCs might be limited by oxygen diffusion to the electrode nanostructured surfaces of biodevices.
Finally, two prototypes for monitoring of glucose and oxygen concentrations were tested using a battery instead of BFCs. From the very beginning of these tests the procedure to calibrate both systems was developed as described in deliverables 5.5 and 5.6. After the calibration of systems, the biodevices were tested. Specifically, the subsequent addition of bioanalytes in physiologically relevant concentrations (5 mM glucose or 0.25 mM oxygen) to the electrochemical cell resulted in the wirelessly measured signal, which was three or more times higher compared to the background value. To conclude, developed wireless electronic and operational units showed very stable and reliable wirelessly monitored signals. The next step was to perform analogous experiments powering the system with a BFC developed by the Consortium. This was done during the final meeting of the Consortium in Bochum (RUB University), Germany. A joint measurement session (5 working days) was organised with several tasks including the final test of two wireless self-powered biodevices, viz. glucose and oxygen sensitive self-contained biodevices. Two compartment BFCs were built using several different (mediator-based and mediator-less) bioanodes and biocathodes because of two main reasons, viz. (i) in order to achieve both current and voltage required for electronics, viz. 44 microampere and 0.57 V and (ii) in order to use all developed bioelectrodes to equalise contribution of all project partners. The following maximal parameters for the combined BFC were obtained: open circuit voltage of 0.73 V, 58 microampere at 0.67 V operating voltage. To increase the power of the BFC, lactose and oxygen at high concentrations, e.g. 10 mM and 0.25 mM, were used as biofuel and biooxidant in the anodic and cathodic compartments of the biodevice. Since lactose and oxygen are required to produce electric power their concentrations could not be significantly decreased during the tests.
The procedure to calibrate systems was exploited and operating voltage in the system was monitored by the external voltmeter. Variation of bioanalytes concentrations resulted in corresponding variation of wireless signals. Moreover, wireless signals from sugar and oxygen sensitive biodevices powered by BFC have been measured successfully for a long time, 1-2 hours. During this time several tens of packages of information were received wirelessly by the receiver, which was located 4 meters away from tested biodevices.
Potential impact:
The multidisciplinary 3D-NANOBIODEVICE consortium addressed several highly challenging, multi-industrial, and interdisciplinary objectives. To solve these,3D-NANOBIODEVICE, brought together leading scientists in the nanobiotechnology field specialising in investigations on enzyme-nanostructure interactions (e.g. enzyme-nanoparticle), integration of nanostructures and enzymes into 3D catalytic and electrically conducting structures for BFC applications and diagnostics. Nanostructures and biocatalysts (redox enzymes), knowledge on their interactions, design of BFCs using nanobiocomposites, and development of modern low voltage miniature electronics were some of the many outcomes of this consortium, outcomes that could not be achieved at any national level. Only European cooperation provided an opportunity to develop 3D nanobiostructure-based bio-electro-catalytic elements, which have a quality required to produce competitive self-contained wireless devices and evaluate these in biomedical application. One of our aims was to put the major fundamental goals of the project a better understanding of the interactions between bioelements and 3D nanostructures and fundamental principles for exploiting and developing electro-conducting nanoarchitectures to assemble highly efficient 3D bioelectrocatalytic structuresinto a practical test by assembling a device consisting of BFC, biosensor, and wireless radio transmitter and evaluating its performance in biological fluids. This was a practical step beyond the state of the art.
A convergence across industries and interdisciplinary studies resulted in breakthrough applications at the interfaces of energy and healthcare areas. The consortium contained two key industrial partners, viz. novozymes A/S (NZ) and Novosense AB (NS), both of which are eager to pick up the technological progress of the project for future commercialisation.
Novozymes A/S, a world leader in industrial biocatalysts production, is part of NovoNordisk, one of the leading producers of insulin and insulin pumps worldwide. The involvement of Novozymes in this project indicates that the company as a whole (i.e. NovoNordisk) is extremely interested, e.g. in acquiring 3D-nanobiodevicetechnology for the development of non-invasive glucose monitoring and wireless actuation of insulin pumps and their commercialisation in the health sector. Additionally, the development of 3D-nanobiobased BFCs is expected to contribute to a breakthrough in the energy area, where novozymes A/S has identified considerable potential to expand their commercial interests as an enzyme producer.
The second industrial partner of the consortium, novosense AB, is a young biomedical engineering company, with currently limited commercialisation potential simply because it is small. However, the company is working with wireless medical devices, which was a key issue of 3D-NANOBIODEVICE project. The company is developing wireless communication protocols to 'wire' medical instrumentation to databases at hospitals. This project enabled the company to develop means of minimising energy consumption for wireless transmission, to design an electric energy harvesting block and voltage amplifier, and thus progress the future integration of BFCs and biosensors into new wireless health care facilities. Additionally, the development of methods of integrating wireless medical devices to databases at hospitals has tremendously shortened the time needed to bring self-contained biodevices to the consumer market.
The potential commercial impact of the project is described in the preceding two paragraphs, which comes through direct collaboration with the industrial partners of the project, NS and NZ. However, the impact directions addressed in collaboration with the partners are much more general. More and more healthcare services and operations are conducted at homes and this trend converts healthcare system into a distributed biomedical industry, where biomedical devices are operated by patients or healthy humans. Healthcare innovations are accentuated by terms such as home based healthcare, mobile healthcare, point of care devices, wireless sensor networks, non-invasive monitoring, wearable and attachable biomedical devices, etc. The expected changes of healthcare systems to some extent can be compared with the changes of financial sector. We use credit cards and decide on credit limits, pay bills over the internet, and take loans without going to the bank. We enjoy financial autonomy. Surrounded by biomedical devices we will decide over our healthcare needs in the future much more than we do today. It is well realised that one of the bottlenecks to the use of distributed biomedical devices is a stable and reliable energy supply. It is hard to accept that we will change batteries in a number of biomedical devices at home every day or even every month. In this project we have been pursuing intensive research to develop and test BFCs as a reliable alternative for powering biomedical devices. We believe that BFCs will become an important power source in new generations of self-contained biomedical devices with self-powering and wireless capabilities. Thus, the progress achieved in this project on the development and combination of BFC with other biomedical devices have a very high impact on innovations promoting the establishment of distributed biomedical industry.
The ultimate goal of the consortium was to create self-contained miniature biomedical devices possessing wireless communication capabilities for implantable exploitations. Taking into account the world market for biosensors (ca. USD 15 billion in 2010 with an annual growth rate of 5%), it is very hard to overestimate the economic effect of this project related on modern biosensor technologies. Over half of the biosensors produced worldwide are employed in glucose meters. The possible economic effect of the project in terms of projected economic feasibility is based on glucose sensitive devices, both in short-term perspectives (i.e. in 3 years after the end of the project; in the year 2015) and in the long run (i.e. in 10 years after the end of the project, e.g. in the year 2022). Moreover, the total economic effect of the 3D-NANOBIODEVICE project is even higher because many other devices with possible commercial futures, e.g. different types of potentially implantable BFCs (mediator based and mediator less), electronic transmitter/transducer systems, oxygen sensitive microbiosensors, was also developed by the consortium. In general, 3D-nanobiodevicebioelectronic technologies, e.g. preparation of efficient 3D nanobiocatalytic structures or separate units, such as BFCs or biosensors, may provide valuable impact for the energy sector (BFCs), the environment (waste water treatment), and the development of in vitro diagnostics, whereas designing of microelectronics and modern software might accelerate the development of the Information technology (IT) sector.
According to the 'World diabetes market' report, diabetes affects approximately 200 million people worldwide and is expected to increase to 300 million diabetics by 2025. In many developed countries (e.g. USA, European Union) more than 5% of the population suffer from diabetes. It is the 6th most common cause of death as recorded on United States death certificates. With a diabetes epidemic underway, there exists strong growth opportunities for diabetes management tools, such as glucose meters. Over the years, glucose-monitoring meters have undergone a sea change, with recent entries featuring wireless and sensor technologies. Sales in United States, the largest market for glucose biosensors, are expected to reach USD 1.3 billion by the end of 2012. The data are based on the prediction that less-complicated devices produced by the consortium, i.e. microbiosensors, will pass clinical trials, be commercialised and account for less than 1 of the total market existing for glucose sensitive biosensors in 2015. Only in 2022 one can expect real commercialisation of wireless implantable self-powered biodevices for continuous glucose monitoring.
It is very hard to estimate the long-term perspectives for devices due to the existence of many different and unpredictable factors affecting their sales. However, taking into account the 5% per annum growth rate on the biosensor market as well as a robust management strategy, one can expect 2.5 times increased sales for microbiosensors in 2022 along with appearance of self-contained implantable biodevices for glucose monitoring on the European market, which will replace simple microbiosensors.
A long-term objective of the Project is to facilitate development of environmentally and body-friendly nanotechnologies for biomedical applications. Profound fundamental knowledge gathered during the project will provide the necessary knowledge for medical exploitation of 3D nanobiostructures as a basis for self-contained wireless biodevices consisting of a BFC, (bio)sensor(s), and a miniature signal transmitter system. The benefits to the society gained from the project can be foreseen as an acceleration of exploitation of knowledge-based products for sustainable innovations of the biomedical industry sector.The project facilitated the integration of biosensors, chips, and BFCs into integrated biomedical devices. It can be predicted that the developed technology will be used to improve quality of life and increase safety in case of widely occurring chronic diseases, when miniaturisation of self-contained and potentially implantable 3D nanobiostructure based devices is done. In the long-run, 3D nanobiostructure-based elements will be essential for constructing devices to be used for neuron/nerve stimulations and compensation of human disabilities.
Additionally, touching on the environmentally and body-friendly development of power sources in form of BFCs, the following can be emphasised. A great deal of the project has been focussed on enhancing BFC performance based on DET reactions between the enzymes and nanostructures. The realisation of DET principle allows more facile construction of BFC and also enables us to avoid additional active component of the system, e.g. mediators, which sometimes are debated as interfering with different redox cycles in living cells. The project has shown that DET based BFC can be as powerful as mediated systems and by this encourages future innovative developments of environmentally friendly DET based devices. This is a very important and fundamental impact arising from the results of this project.
All scientists participating in this project were exposed to a broad spectrum of both fundamental and technological knowledge and, equally important, they were trained in human resource management, gender and ethics related problems, importance of public acceptance of advanced science/technology, and a business/IP-minded thinking. As the final result, in the second part of the project (month 18-36) a close dialogue with two companies, which are established in the relevant sectors (Energy and biotechnology sectors), was organised by partner Mah with the support from MINC (www.minc.se) and Medeon (www.medeon.se).
The fundamental and technological knowledge developed during the project was discussed with CAVAC AB (www.cavac.se). Specifically, this young and ambitious Swedish company is interested in designing efficient and stable BFCs for different applications. Partner Mah was contacted by CAVAC as a possible party, which might provide technology to design efficient and stable cathodes. However, due to the lack of patents in this particular area (the Project was focused on biodevices instead of devices based on inorganic or organic catalysts) and financial constraints of the SME, no substantial agreement has been achieved so far.
Another example of commercialisation efforts from 3D-NANOBIODEVICE are in negotiations between the Finish company, VTT Biotechnology (www.vtt.fi) and Mah concerning a possible transfer of technology developed by researchers at Malmoe University during the project, specifically flexible, stable and efficient BFCs for extra vivo applications. Negotiations are ongoing and based on the patent WO/2011/117357 entitled: 'Flexible BFC, device and method'obtained by Mah with the financial support from 3D-NANOBIODEVICE.
Communication with the scientific community and the public at large was also an important task of 3D-NANOBIODEVICE. Accumulation of knowledge and its faithful dissemination to the society fully supports the ECs policy on integrated, safe, and responsible nanotechnology. The consortium hopes that these activities during the project enabled to increase both confidence and trust in the EC as a truly transparent and trustworthy communicator.
In addition to classical dissemination roots (publications in peer reviewed journals, conferences, workshops, symposiums, congresses, and public presentations) and novel/electronic means (web), results from the project were used in lectures, education programs, and other communication materials (newsletters, flyers, posters) via mass communication media. Detailed results on publications, patents, and disseminative activities of the consortium are presented in the final report, as well as summarised in Table 6.
List of websites: www.mah.se/3dnanobiodevice.