The project aims at developing novel biosensors based on nanostructured materials for self-powered biodevices assembly. Biofuel cells (BFCs) is an emerging technology because of their ability to directly generate electricity from biologically renewable catalysts and fuels. Due to the boost in nanotechnologies, significant advances have been accomplished in BFCs. Although it is still challenging to promote the performances of BFCs, the adoption of nanostructured materials for BFC construction represents an effective and promising strategy to achieve high energy production.
Among such nanomaterials, gold and silver nanoparticles, carbon nanotubes and graphene will be particularly studied. A careful characterization of the nanomaterial will be carried out by using several techniques such as scanning electrode microscope, transmission electron microscopy, energy dispersive spectroscopy, dynamic light scattering and UV-Vis spectroscopy.
The project envisaged the realization of enzymatic electrochemical biosensors for the sensitive detection of glucose, alcohol and fructose. These analytes will be detected with nanomodified second and third generation biosensors realized with the use of a redox mediator (MET) and with a direct electron transfer reaction between enzyme and electrode (DET), respectively.
The kinetic parameters of the corresponding biosensors will be carefully evaluated.
The results arising from the study of the biosensors will be useful to set up a technological platform for the future development of portable, cheap, fast, highly sensitive self-powered biosensors based on enzymatic biofuel cells for several application fields.
The final outcome of this project will be the design and development of self-powered biofuel cells by introducing new nanotechnology-based devices, thus reducing costs and increasing the competitiveness in terms of sensitivity, time of analysis and fields of applicability.
The core of innovation of this project is the investigation of several nanostructured materials for electrode modification in order to increase the sensitivities and to lower the detection limits of biosensors for analytes of clinical and food interest, such as glucose, alcohol and fructose.
We will develop different electrochemical biosensors based on single-wall and multi-wall carbon nanotubes and on chemically derived graphene, including graphene oxide and reduced graphene oxide, properly functionalized with metal nanoparticles (NPs) for improved performances. We will investigate and discuss different strategies to prepare the nano-modified electrodes.
In our project, we will develop a new green and sustainable biosynthesis of gold and silver nanoparticles (AuNPs and AgNPs). Green synthesis is an arising field in nanotechnology sustainable chemistry for the preparation of metallic nanoparticles. Instead of using a synthetic chemical reducing agent, which may be harmful to the environment, a plant or fruit extract will be used as the reductant. The use of these natural materials offers low cost, reduced toxicity and low temperature, pressure and energy requirements. An important goal of our proposal is to eliminate the use of toxic reducing agents such as sodium borohydride and their reaction products during the synthesis of AuNPs and AgNPs and to provide a more sustainable, rapid, cheap, eco-friendly green synthetic method from HAuCl4 and AgNO3 using extracts of plants and fruits. Our synthetic method will be carried out at room temperature and does not require expensive reagents or instrumentation and skilled personnel.
Particular attention will be paid to the optimization of the communication between the redox protein and the sensing transducer by controlling the biorecognition element coverage on the electrode surface and by using suitable functional linker groups. The biorecognition elements will be selected and characterized in order to acquire enough knowledge about their interaction with the analytes to be investigated. Particular interest will be focused on the use of engineered enzymes.
Cellobiose dehydrogenase and fructose dehydrogenase will be engineered in order to change some of their physicochemical properties (e.g. deglycosylation) to improve the biosensors capacitance to increase the faradaic efficiency or the substrate specificity.
In particular, structural comparisons of the cellobiose dehydrogenase (CDH) active site and that of glucose oxidase (GOx) highlighted a conserved Cys residue in the CDH active site, in the carbohydrate-binding pocket, while a Tyr residue is found at the equivalent position in GOx (at position 68). This single Cys to Tyr mutation in the active site of CDH will be applied to the Class II CDHs Corynascus thermophilus (CtCDH) and its mutant CDH C291Y. These enzymes show optimal electrocatalytic properties for DET at neutral pH, a fundamental characteristic for biosensors and enzymatic fuel cells.
Fructose Dehydrogenase (FDH; EC 1.1.99.11) from Gluconobacter japonicus NCBR 3260 will be engineered by encoding each subunit of the FDH complex from G. japonicus NBRC3260 and constructing an expression system to highly produce FDH in a Gluconobacter oxydans strain, changing the translational initiation codon from TTG to ATG (from FDHTTG to FDHATG). FDHATG is a heterotrimeric membrane-bound enzyme complex with a molecular mass of ca. 140 kDa, consisting of three subunits. Within this project, we would like to further engineer the enzyme by cleaving the amino acid sequence on the N-terminus of subunit II and the Heme c moieties one by one to reduce the over-potential required to catalyze the oxidation of fructose. The downsizing protein engineering is supposed to cause an increase in the surface concentration of the electrochemically effective enzyme and an improvement in the heterogeneous electron transfer kinetics.
Another important innovative aspect of this project is the choice of the proper redox mediator in the case of the realization of mediated electron transfer based biosensors. We will study and characterize different Osmium complexes which are interesting because it is possible to tune their redox potential by varying the functional groups bound to the heme moiety.
Also the use of screen-printed modified electrodes instead of classical graphite electrodes represents an innovative point of the project. Screen-printed electrodes are suitable for working with microvolume and for decentralized assays.
Moreover, the approach presented in this project for the development of new platforms for cheap and highly sensitive self-powered biosensors based on EBFCs could offer significant advantages over current methods and could afford the diagnosis in underdeveloped and developing countries, helping to open the door to global access to the diagnosis.