Final Report Summary - BIOMIMIC (Biomimetic sensors as new generation of biotechnological devices for food safety and quality monitoring)
Project context and objectives
Between 2009 and 2012, 14 researchers from three different institutions and countries have participated in this international research exchange project (four Italians from the University of Teramo, five Czechs from Masarykova Univerzita and five Brazilians from Sao Paulo State University, Institute of Chemistry, Department of Analytical Chemistry). During this period, more than ten publications were or will be published in peer-reviewed journals and over 20 works presented at international congresses by invited speakers, or as oral or poster presentations.
The present research work was driven by the need to eliminate a serious problem in the international community and aiming to substitute biological receptors with an easier and more user-friendly sensing system for use in food-safety and quality monitoring. The strategic importance of the research was derived from expertise that is not available in any individual country, thus solving the problem with global rather than just European dimensions. The potential biosensors or biomimetic sensors, either singly or in combination as an array, and pattern recognition elements could revolutionise applications within the EU and on the world market.
Work performed
The key point of this research was to take advantage of molecular modelling software to rationalise the design of molecular traps with the aim of reducing by orders of magnitude the efforts necessary to obtain specific receptors. The basic idea was to design peptide-based structures matching specific requirements by mimicking real biological molecules in terms of shape and chemical properties.
Main results
The selection of targets with solved structures and reported affinities from literature was evaluated. Pesticides, cocaine, thrombin, dioxins and polychlorinated biphenyls (PCB) receptors with a tri-dimensional structure were selected. Parameters related to the interaction between receptors and targets were studied by using existing programmes for molecular structure analysis and visualisation, such as Hyperchem, VIDA from OpenEye Scientific Package, RasMol, SwissPDB Viewer, Chimera and others.
The transduction technologies and approaches for biomimetic devices was evaluated and carried out. An especially portable instrumentation, Bioca (electrochemical impedance spectroscope), was evaluated to use within the project and compared with the advanced impedimetric system Autolab. Gold-based screen-printed electrodes with several sensing channels were delivered to become modified with biomimetic receptors and evaluated for the affinity-binding with the target. All supporting tools, software, sensors and reagents for the experimental part were arranged and tested.
Small sequences of peptides were designed and synthesised. The peptides were prepared on the principle of 'solid-phase' synthesis. A theoretical amount of peptides was calculated by obtaining, after the lyophilisation step, a peptide purity of 96.9 %. Impedance measurements were performed using CDtrode technology where peptides were covalently immobilised before preparation and cleansing. Different analytical parameters were optimised, such as a pH and ionic strength buffer, before a carbaryl/peptide interaction was analysed in standard solution. Cross reactivity was calculated using gluthation. Moreover, a comparison of CDtrodes and screen-printed electrodes was evaluated.
The surface of different CDs was analysed by AFM in order to understand the problem of repeatability in the receptor's immobilisation on a CDtrode surface.
A computationally assisted method to study aptamer-protein interaction was evaluated with the aim of streamlining the screening and selection of new aptamers. Receptors were compared using signal amplitude, sensitivity (slope), linearity (R2) and reproducibility (CVav %). The experimental results were in agreement with the simulation findings.
A post-PCR nucleic acid work by comparing experimental data, from electrochemical genosensors, and bioinformatics data, derived from the simulation of the secondary structure folding and prediction of hybridisation reaction, was carried out in order to rationalise the selection of single-stranded deoxyribonucleic acid (ssDNA) probes for the detection of two Bonamia species, B. exitiosa and B. ostreae, parasites of Ostrea edulis.
The oligopeptide proposed by molecular modelling (NH3+-Cys-Glu-His-Gly-Gly-Pro-Ser-COO-) was synthesised by solid-phase peptide synthesis methodology and characterised by high-performance liquid chromatography and mass spectrometry, which showed a strong interaction with the pesticide dichlorvos (K = 4,10 x 105 M-1) in spectrophotometric studies.
Using quartz crystal microbalance (QCM) it was possible to achieve real-time detection of antigen-stimulated exocytosis of RBL mast cells. The monitoring of exocytosis was performed in the flow cell and the stimulation of cells was done using a flow injection system (FIA), proving that it can be used as a tool for the rapid screening of pharmaceutical substances as the potential inhibitors of exocytosis.
The exchange between the partner countries allowed the development and implementation of biosensors, which provided a tremendous impact, both as on-site, automated monitoring systems, and also as hand-held, miniaturised instrumentation for on-site control. These systems were cheaper than the conventional analysis tools, and they can be used for screening purposes. The biosensors obtained in this research were used in the field of food control and analytical assays showing very promising results, both theoretically and experimentally, which may ultimately lead to an improved quality of life for all.
The new techniques and approaches resulting from this research may alter our ability to monitor and predict susceptibility to and effects of infection. Depending on the biological recognition element used, biomimicry can be used as a tool to screen for different food contaminants, which can range from pesticide residues to genetically modified organisms (GMOs). Standardised technologies were developed and implemented throughout European countries, including associated candidate third countries. This research project also combined interdisciplinary aspects by using chemistry, biology and informatics. Areas such as quantum chemistry, statistical thermodynamics, analytical chemistry and biochemistry were implied in this research.
Between 2009 and 2012, 14 researchers from three different institutions and countries have participated in this international research exchange project (four Italians from the University of Teramo, five Czechs from Masarykova Univerzita and five Brazilians from Sao Paulo State University, Institute of Chemistry, Department of Analytical Chemistry). During this period, more than ten publications were or will be published in peer-reviewed journals and over 20 works presented at international congresses by invited speakers, or as oral or poster presentations.
The present research work was driven by the need to eliminate a serious problem in the international community and aiming to substitute biological receptors with an easier and more user-friendly sensing system for use in food-safety and quality monitoring. The strategic importance of the research was derived from expertise that is not available in any individual country, thus solving the problem with global rather than just European dimensions. The potential biosensors or biomimetic sensors, either singly or in combination as an array, and pattern recognition elements could revolutionise applications within the EU and on the world market.
Work performed
The key point of this research was to take advantage of molecular modelling software to rationalise the design of molecular traps with the aim of reducing by orders of magnitude the efforts necessary to obtain specific receptors. The basic idea was to design peptide-based structures matching specific requirements by mimicking real biological molecules in terms of shape and chemical properties.
Main results
The selection of targets with solved structures and reported affinities from literature was evaluated. Pesticides, cocaine, thrombin, dioxins and polychlorinated biphenyls (PCB) receptors with a tri-dimensional structure were selected. Parameters related to the interaction between receptors and targets were studied by using existing programmes for molecular structure analysis and visualisation, such as Hyperchem, VIDA from OpenEye Scientific Package, RasMol, SwissPDB Viewer, Chimera and others.
The transduction technologies and approaches for biomimetic devices was evaluated and carried out. An especially portable instrumentation, Bioca (electrochemical impedance spectroscope), was evaluated to use within the project and compared with the advanced impedimetric system Autolab. Gold-based screen-printed electrodes with several sensing channels were delivered to become modified with biomimetic receptors and evaluated for the affinity-binding with the target. All supporting tools, software, sensors and reagents for the experimental part were arranged and tested.
Small sequences of peptides were designed and synthesised. The peptides were prepared on the principle of 'solid-phase' synthesis. A theoretical amount of peptides was calculated by obtaining, after the lyophilisation step, a peptide purity of 96.9 %. Impedance measurements were performed using CDtrode technology where peptides were covalently immobilised before preparation and cleansing. Different analytical parameters were optimised, such as a pH and ionic strength buffer, before a carbaryl/peptide interaction was analysed in standard solution. Cross reactivity was calculated using gluthation. Moreover, a comparison of CDtrodes and screen-printed electrodes was evaluated.
The surface of different CDs was analysed by AFM in order to understand the problem of repeatability in the receptor's immobilisation on a CDtrode surface.
A computationally assisted method to study aptamer-protein interaction was evaluated with the aim of streamlining the screening and selection of new aptamers. Receptors were compared using signal amplitude, sensitivity (slope), linearity (R2) and reproducibility (CVav %). The experimental results were in agreement with the simulation findings.
A post-PCR nucleic acid work by comparing experimental data, from electrochemical genosensors, and bioinformatics data, derived from the simulation of the secondary structure folding and prediction of hybridisation reaction, was carried out in order to rationalise the selection of single-stranded deoxyribonucleic acid (ssDNA) probes for the detection of two Bonamia species, B. exitiosa and B. ostreae, parasites of Ostrea edulis.
The oligopeptide proposed by molecular modelling (NH3+-Cys-Glu-His-Gly-Gly-Pro-Ser-COO-) was synthesised by solid-phase peptide synthesis methodology and characterised by high-performance liquid chromatography and mass spectrometry, which showed a strong interaction with the pesticide dichlorvos (K = 4,10 x 105 M-1) in spectrophotometric studies.
Using quartz crystal microbalance (QCM) it was possible to achieve real-time detection of antigen-stimulated exocytosis of RBL mast cells. The monitoring of exocytosis was performed in the flow cell and the stimulation of cells was done using a flow injection system (FIA), proving that it can be used as a tool for the rapid screening of pharmaceutical substances as the potential inhibitors of exocytosis.
The exchange between the partner countries allowed the development and implementation of biosensors, which provided a tremendous impact, both as on-site, automated monitoring systems, and also as hand-held, miniaturised instrumentation for on-site control. These systems were cheaper than the conventional analysis tools, and they can be used for screening purposes. The biosensors obtained in this research were used in the field of food control and analytical assays showing very promising results, both theoretically and experimentally, which may ultimately lead to an improved quality of life for all.
The new techniques and approaches resulting from this research may alter our ability to monitor and predict susceptibility to and effects of infection. Depending on the biological recognition element used, biomimicry can be used as a tool to screen for different food contaminants, which can range from pesticide residues to genetically modified organisms (GMOs). Standardised technologies were developed and implemented throughout European countries, including associated candidate third countries. This research project also combined interdisciplinary aspects by using chemistry, biology and informatics. Areas such as quantum chemistry, statistical thermodynamics, analytical chemistry and biochemistry were implied in this research.