THE PROJECT

1. Summary

The aim of this project is to design different next generation libraries of biosensors that will yield ready-to-go analytical tools against any target in unprecedented speed and high affinity. The project intends to focus on aptamers, a new generation of recognition elements that will be integrated into bioelectronics devices.

The huge number of candidates to be tested using a combinatorial trial and error approach will be radically reduced by rationalizing the strategy to design the aptamers. A new virtual screening process will be used for the generation of aptamers by the help of modern molecular modelling tools. The design and binding of large database will be created and screened by adding or mutating molecule bricks (amino acids and DNA), minimizing experimental tests and therefore reducing the research costs.

These sensor elements will be organized in medium to high density nucleic acid and peptide arrays with different transducing principles. In particular, we will design and produce an electrochemical sensor array platform based on screen printing technology together with suitable extraction- and pre-concentration procedures by coupling aptamers to nanoparticles.

The analytical procedures produced will be tested using first standards and then real samples. The target molecules will be analytes related to health and safety control (Xenobiotics: e.g. PCB, pesticides. Mycotoxins: e.g. patulin, nivalenol, Antioxidants: e.g. resveratrol, luteolin).

The proposal has strong focus on development of concrete evolutionary new biosensors for application to diagnostics, food, terrorism threat agents, environmental and new frontiers analysis such as bioanalysis in the space.

2. Excellence

2.1 Quality, innovative aspects and credibility of the research (including inter/multidisciplinary aspects)

· Introduction, state-of-the-art, objectives and overview of the action

The analytical chemistry community continues to search for portable analytical techniques that can give reliable, on-site results for a variety of matrices and a host of analytes. The elaboration of biosensors is probably one of the most promising ways to solve some of the problems concerning sensitive, fast and cheap measurements.

The rapid development of biosensors for biochemical analysis has been greatly promoted by the progress of microfabrication techniques and microchemical systems and the use of these devices have attracted much attention of scientists and engineers. Recent advantages in nanobiotechnologies (surface functionalization and patterning, detection, microfluidics, integration) tools will have a decisive impact on the performance of the new generation of biosensors and biochips .

Generally, in analytical assays, when the detection system requires a bio-molecular recognition event, antibody-based detection methodologies are still considered the standard assays in environmental, food and clinical analysis. These assays are well established and they have been demonstrated to reach the desired sensitivity and selectivity. However, antibodies are typically large, multichain molecules that require post-translational modifications and disulphide bond formation for their stability. Their immobilisation on surfaces leads to significant cross-reaction with proteins other than those to which they were selected to bind. Further, antibodies are generally fragile against physico-chemical stresses (pH, ionic strength, solvents, etc.) reducing the shelf life and reliability.

In order to circumvent some of these drawbacks, other recognition molecules are being and have to be explored as alternatives. Recently, attention has turned toward reagents that may be considered as artificial antibodies, known as aptamers, made from short single strands of DNA or oligopeptides that adopt specific three-dimensional conformations allowing biorecognition of specific target (small or large molecules).

Aptamers have started to be used in many analytical applications, such as chromatography, electrophoresis, mass spectrometry, molecular beacons, sensors and biosensors. In particular, aptamers have become increasingly important molecular tools for diagnostic and therapeutic.

· Research methodology and approach

The aim of this project is to investigate the chemistry of the aptamers, the range of possible three-dimensional structures which an aptamer can fold into, as well as the stability and analytical performances of aptasensors. This exceptional propensity of aptamers to assume an array of secondary (and tertiary) structural motifs with different shapes will be also studied by using modern molecular modelling tools.

In practice, APTASENS comprises of the following steps:

· Originality and innovative aspects of the research programme

The aptasensors will be tested in different target concentration, performing calibration curves; also selectivity will be evaluated. Then, contaminated real samples will be analysed. Quite often, available bioreceptor elements are sensitive to harsh conditions, e.g. cell based systems that need a physiological pH. This often leads to the realisation of “academic” demonstration and very few applications in real samples. We consider this point being one of the major drawbacks of the biosensor area. We want to overcome this drawback with the help of a panel of different nucleic acid and peptide aptamers that all bind specifically to a given analyte (multivariate analysis). We will validate our procedures in natural waters, waste waters, soil, food and biological samples by comparing analytical performances of aptasensors with traditional methods.

2.2 Clarity and quality of transfer of knowledge/training for the development of the researcher in light of the research objectives

The training program for acquiring additional scientific and complementary skills is described in this agenda:

-Daily. The researcher work will be supported and super vised for step by step working day by scientists in charge and experienced researchers team of the outgoing and/or return institution. The researcher will tutor different PhD and MsC students supporting research laboratory and/or bioinformatics procedures and helping in daily work. The website project will be upgrade daily for remote assistance, sharing and comments.

-Weekly. A meeting group once a week will be scheduled for discussing and supporting the researcher project. The accomplishment of the research objectives, new objectives established during the course of work and new lines of research mentioning also unsuccessful approaches and unforeseen developments along with scientific and technical aspects problems will be resumed and discussed with the host team.

-Monthly. The researcher will participate to specific classes or/and seminars taking by international visitors usually invited monthly by host institution. Eventually the researcher will actively participate to international congresses exposing the project results progress in oral and poster presentations. Once a month the researcher will follow specific classes, also using remote assistance, for reinforcing writing proposals, research management, communication, leadership and teaching skills.

Moreover Training to the researcher will be done using specific classes taking by experts of host team or international invited speakers, specialized in different fields

-Yearly. Every year the outgoing team organizes important international congresses on theoretical and applied chemistry where key scientific personalities and commercial companies take part. This is an important chance for the researcher at any level from the organization of international events to the meeting and data interchanging of international celebrities in chemistry meeting congresses.

Every year to proof the training effectiveness of the project, the researcher is expected to produce at least 4 publications in international peer-reviewed journals, 1-2 project proposals and 1-2 teaching courses.

2.3 Quality of the supervision and the hosting arrangements

The Spanish and North American supervisors UCM and UCSD), have been pioneers and leaders in biosensors field from genetically engineering living cells to nanomotors resoling problems of molecular sensing, diagnostics, drug delivery, etc. They are also in the editorial board of several scientific international journals with high impact factor (Electroanalysis, Talanta, Analytica Chimica Acta, Biosensors and Bioelectronics, ACS journals etc.) The have provided real-life solutions to important scientific problems.

Prof Wang (UCSD) will act as outgoing host institution supervisor and will take responsibility for data planning and the successful and widespread broadcasting of the results of the project. Prof Wang will be responsible for the assessment and management of the scientific knowledge and technology created by the project. The expertise and excellence of Prof Wang is recognised around the world resulting among the 100 most influential people in Analytical Sciences (2013) and making the Top 10 Most Cited Chemists in the World. This important group will be the responsible of the 2/3 of the project guiding the dissemination and exploitation of results and will contribute within and between all work packages. The activity of nanoenginering group (UCSD) focuses on the fields of nanobioelectronic which is a rapidly developing field aimed at integrating nano- and biomaterials with electronic transducers. This highly multidisciplinary research combines fundamental studies with forward-looking engineering efforts.

Prof. Pingarron (UCM) will act as return host institution supervisor and take responsibility for identifying and solving technical problems across the work packages. He will provide the management of the technical progress between WPs and towards the objectives, as well as the exchange of results and knowledge between the universities and EU. Prof. Pingarron has a large experience in biosensors field In particular the return phase group has wide experience on bio-structured devices and biosensors, especially using engineered biological structures, thus ensuring innovation and a high degree of success in achieving the objectives of the proposed work.

The project partners lab are full equipped with all necessaries offering working conditions to realise the potential of individuals and to provide new career perspectives. These modern laboratories are well equipped with an advanced analytical instrumentation and microfabrication tools, along with computing clusters for intensive calculations required in virtual process totally accessible also via web. These include modern electrochemical (voltammetric, galvanostats, impedance) analyzers, potentiometric analyzers, lab-on-chip systems, high-precision screen printer (MPM), spin coater, plasma cleaner, advanced optical microscopes (for nanomotor tracking and microfabrication efforts) and a graphic station for advanced microfabrication, Chromatography facilities (HPLC, GC, etc.); surface characterization with SEM and other microscopic techniques; spectroscopic equipment with nuclear magnetic resonance (NMR), mass spectroscopy (MS).

2.4 Capacity of the researcher to reach and re-enforce a position of professional maturity in research

The researcher knowledge will increase in efficiency for designing biomimetics and screening new applications to improve biosensors area. This proposed research will bring to the applicant the expertise of the scientists with large experience in bioinformatics and biosensors area. Both outgoing and host institution teams will train the applicant for the development and the use of theoretical and applied chemistry tools, in order to acquire new knowledge in sensing strategies at international level.

This research will be the junction for the flow of the academic knowledge to the real applications; it will be both a research experience and a school for the development and use of new bioinformatics methods proofed by sensors technology by producing analytical tools for analytical detection helpful in resolving many health and safety real problems.

This project will be both a research experience and a school for the development and use of new modeling techniques and computational approaches in biosensors field, bringing International collaboration closer different nationalities, skills, culture and trainings. Moreover the interdisciplinary partnerships will be the way for new and interesting ideas to face a real scientific problem.

This project will contribute to the applicant’s career development with transfer of expertise into analytical and theoretical chemistry having important added values to:

1. Produce rational candidates via molecular modeling, bioinformatics tools for large database screening 2. Comprehensive generation of receptors for a particular target, practical solution of real laboratory problems. 3. Improving laboratory skills in manufacturing biosensors. 4. Understanding existing and creation of new sensing methods. 5. Fundamental knowledge required to produce practical, competitive analytical devices. 6. Enhanced communication between scientists, engineers, end-users and public.

3. Impact

3.1 Enhancing research- and innovation-related human resources, skills, and working conditions to realise the potential of individuals and to provide new career perspectives

The Researcher, as well as institutions involved in the project, can take advantages from sustainability together with the economic impact of the deliverables identified in the following areas:

- Environmentally friendly analysis systems. In comparison to conventional analysis, these systems need only minor amounts (or no amounts) of organic solvents and will therefore contribute to a sustainable analytical industry.

-Rationalization of protocols to select molecules avoiding trial and error and/or combinatorial approach, the virtual approach is critical to provide new skills and career perspectives of seconded researchers with specialization in computing methods linked to chemistry, multivariate manipulation of databases for molecular systems and post processing data.

- Development of methods, programs and databases applied to biology, molecular modelling field, protein structure, their interfaces in complexes oriented to obtain predictive criteria for protein compounds and/or aggregates.

- Miniaturisation will lead to lighter systems, which will be easier to ship and to carry to different places, thus increasing the mobility of analytical systems (e.g., on-site analysis at places, where there is no lab nearby).

- The usage of synthetic DNA and peptide recognition elements leads to higher selectivity and reproducibility, such reducing the amount of sample preparation and the sample size. This enables to ship samples much easier and less storage space is needed.

- Generally, these systems are fast and enable the user to measure/screen more samples per time.

The development of miniaturised, single analyte and multianalyte sensor systems is of worldwide interest, where Europe should play a leading role to enforce its own policies and through this will set global standards.

On the other hand, there is no question that the application of aptasensors in the field of analytical chemistry will unleash such a vital influence that will have an enormous impact on our daily lives by altering our ability to monitor and predict susceptibility to and effects of control, hence leading to an improved quality of life for all.

3.2 Effectiveness of the proposed measures for communication and results dissemination

The dissemination of the scientific results of the project will be achieved through publications of the results in scientific journals.

To ensure the protection of intellectual properties all publications will be evaluated by both supervisors under the guidance of the EU manager before publication.

We would like to bring new technology not only to specialists but also to introduce into these new fields of research the students of secondary, therefore the new technologies will be presented at open door days that are planned in “Implementation” section. This project will enable researcher to bring more closely the business world to academic by demonstrating the use of aptamers in a new field of analytical properties, increase knowledge about unwanted bioactivity and data on the relation between structure and binding as well as structure and function relation.

The Dissemination and Exploitation Plan includes:

-coordinating an agreement which satisfactory for the participants

-contract and formalising exploitation restrictions, licensing arrangements, protection of results and methods of disseminating results.

-conduct reviews of the project’s impact on economic, ethical, gender and societal issues.

The Dissemination and Exploitation channels and formats between the partners will be established though a project web-portal provided by APTASENS to ensure rapid and robust transfer of information, results, data, dialogue, reports.

A monthly report regarding dissemination and exploitation of the project results will be collated every six months and sent to the EU Project Technical administrator. The reports will cover progress on each of the active task and the actions needed for the progress of the project.

4. Implementation

4.1 Overall coherence and effectiveness of the work plan, including appropriateness of the allocation of tasks and resources

During the project researcher will be involved in modern molecular modelling development, aptamers chemistry, electrochemical methods optimization, multiple-data automation and elaboration as well as practical testing of small density array of aptamer receptors. Below the work packages described one by one reporting objectives, tasks and deliverables.

Work Package 1 Analysis of structural motifs with modern molecular modelling tools

Objectives: O1.1. Selection and customization of molecular modelling software. O1.2. In silico screening of large libraries of compounds. algorithms for the prediction of secondary structures. O1.3. Improving affinity in successive screening rounds by permutating the oligonucleotide and peptide sequences at some positions and examining the resulting affinity for the target molecules.

Tasks: T1.1. Combination of molecular modelling methods and surface generation algorithms in a program module T1.2. Characterization and interaction of target and receptor including chemical environment phenomena. T1.3. Generation of rationalized libraries of receptors, by modifying the structures of input molecules, T1.4. Docking methods employ scoring functions to assign a binding score.

Deliverables: D1.1. Dataset of aptamer receptors (eventually chemically modified) binding ligands in different chemical environments. Protocols for rationalizing the choice of selective receptors avoiding trial and error and combinatorial approach. D1.2. Software customization for generating optimized ligands library. Validation with reference data D1.3. Calculation of solvent impact on complex stability and fast molecular screening of large datasets of structures. D1.4. Computational method to calculate confident association free energy in small vs. small molecule complexes using customized methods at variable precision degree

Work Package 2 Chemistry of the aptamers: Immobilisation and stability

Objectives: O2.1. Exploring routine process synthesis performed automatically in any amount and in various chemical modifications O2.2. Testing different strategies to chemically synthesize aptamers functionalised with convenient molecules for signal transduction or to improve stability (by using proper modifications and cleaning procedures) O2.3. Producing aptamers as attractive reagents for use in the therapeutic and other applications where quality control is critical. O2.4. Investigating new solid support materials like nanotubes and/or nanoparticles as immobilization platform. O2.5. Optimising the performance to immobilise aptamers onto electrochemical surfaces assembly (size, stability, materials, etc.), in order to increase reaction signals and improve detection limits.

Tasks: T2.1. Performing different solid support synthesis procedures using nanotubes or/and nanoparticles as sorbent materials. T2.2. Selecting reagents with lower toxicity and higher bio compatibility to be integrated in aptamer synthesis. T2.3. Evaluating different immobilisation protocols for functionalising aptamers with molecules to improve electrochemical signals and stability. T2.4. Coupling aptamers with electrochemical surfaces. Testing different modification surface strategies. Investigating new sensor surface materials.

Deliverables: D2.1. Protocols for different synthesis of aptamers functionalised with convenient molecules for increasing signal and stability D2.2. Procedures for combining aptamers with new materials like nanotubes and nanoparticles. D2.3. Know-how for coupling functionalised aptamers with electrode surfaces and inks (graphite, silver, gold, platinum). D2.4. Seminars, communications and reports when requested as well as scientific publications

Work Package 3 Optimisation of electrochemical methods based on aptamers

Objectives: O3.1. Optimising the performance of the experimental assembly based on aptamers by means of integrated and miniaturised instrument for rapid (seconds or minutes) determination of targets, in the range of ppm to ppb, with disposable electrochemical technology. O3.2. Developing predictive models to assess the behaviour of aptasensors in the detection of targets. Appling statistical analysis of the data obtained with the proposed system in order to define the sample contamination level. O3.3. Showing practical applicability of electrochemical techniques with the appropriate stability, sensitivity, selectivity, robustness, quantification of sensor signals, and affordable price. Testing either single analyte or multi-analyte determinations, in solution or immobilized onto the transducer surface.

Tasks: T3.1. Studying different electrode materials and inks. T3.2. Optimizing electrochemical procedures for different class of compounds. T3.3. Design and development of the assays, in terms of testing and optimization steps. T3.4. Developing software prototype running on Pocket PC or PC in order to allow the application of different pulse voltammetry. T3.5. Evaluating both equilibrium and kinetic processes to realize bio-chemical assays with characteristics of selectivity, sensitivity and long term stability and storage.

Deliverables: D3.1. Protocols for different electrode surfaces and different materials and inks (graphite, silver, gold, platinum).Equilibrium and kinetic processes of electrode surfaces. D3.2. Prototype and characterisation of multi-parameter electrochemical biochip implemented to be used in the final tests, including all characterization and validation data, especially with respect to regeneration and reusability D3.3. Structural (stability) and functional (specificity) characterisation of aptasensors. D3.4. Electrochemical methods for miniaturized and portable instrument based on aptasensors.

Work Package 4 Improvement of small density electrochemical aptasensors array

Objectives: O4.1. Improving and testing small density array of electrodes modified with aptasensors in order to approach the sensitivity and reproducibility of the classical analytical methods O4.2. Support of miscellaneous measurement protocols (e.g. direct calibration-based measurement or standard addition-based measurement). O4.3. Multiple level fusion of data so as to increase measurement accuracy/range and reliability. Multivariate analysis for post processing data elaboration O4.4. Providing a set of final protocols and platform demonstrations with acceptance of test protocol by all concerned parties (instrument manufactures, suppliers and relevant authorities) to be presented as a European standard.

Tasks: T4.1. Evaluation of small density array aptasensors and their analytical application to real samples T4.2. Reducing non-specific binding in case of interferences from food matrices. T4.3. Producing fast, low-cost and innovative methods especially designed for in situ analysis without or with less pre-treatment and purification steps. T4.4. Performing and optimising the whole analytical process, in a single analytical procedure. T4.5. Construction of a large database of sensor responses. Database and measurements are essential for both pattern recognition and robustness of the identification methods. T4.6. Providing data supporting modelling analysis Construction of a large database of sensor responses versus virtual response. T4.7. Comparison with reference methods for chemical as well as biochemical analysis

Deliverables: D4.1. Database of small density array results. Technical description of experimental performances versus simulated one. D4.2. Assays methods including all characterization and validation data, especially with respect to reference methods for chemical as well as biochemical analysis. D4.3. Simultaneous detection of multi-analyte using both qualitative and quantitative approaches involving the coupling of small density electrochemical array aptasensors and advanced chemometric data processing.

4.2 Appropriateness of the management structure and procedures, including quality management and risk management

Formal communication channels and formats will be established though a project web-portal provided by APTASENS to ensure rapid and robust transfer of information, results, data, dialogue, reports, audit certificates, and cost claims between the partners. Deliverables, resources and costs will be monitored and managed through the project portal via monthly reports from the researcher and supervisors. The monthly reports will be collated every six months and researcher will transfer to the EC Project Technical Officer. The report will cover the progress on each of the active task and the actions needed for the following six month period.

Supervisors will take responsibility for the overall financial, contractual, legal and administrative aspects of the project’s management as well as all communication with the EC including:

- Collation of all deliverables and reports; - The overall legal, contractual, ethical, financial and administrative management of the project; - Preparing, updating and managing the consortium agreement between the participants; - Resolution of any administrative or contractual issues within the partnership and with the commission, - Organisation of project technical management and exploitation board meetings, - Overseeing the promotion of early stage and experienced researcher along with gender equality in the project; - Collation of all the cost statements, - Ensuring prompt payments of financial contributions, - Obtaining audit certificates (if any) by each of the participants, - Coordinating payments and the distribution of money

A Risk & contingency management will be also enabled through the application of Failure Mode Evaluation & Analysis techniques to individual Work Packages as well across the interdependencies between WPs. Specific potential failure modes will be identified together with an assessment of risk and a layered remedial action strategy to: 1. Reiterate elements of the task whilst varying the activity in a manner likely to improve the result. 2. Compensate for lower levels of failure by an adjustment in related deliverables 3. Adoption of an alternative development route.

4.3 Appropriateness of the institutional environment (infrastructure)

Outgoing and return partners have a sensor development group that works in the labs of their own Department at the academic level (University). The groups are specialised in the development of biosensors using electrochemical, optical (electrochemiluminescence, fluorescence, fibre optics) and piezoelectric (QCM) transducers, therefore the lab are full equipped with all necessaries for: - Immunoassay or enzyme assay development and characterization; -Screen printing for mass production of electrodes; -Self-assembled monolayers for biocomponent immobilization; -Electropolymerization for biocomponent immobilization; -Development of novel bioconjugation techniques; -Development of novel immobilization techniques; -Affinity chromatography for antibody purification; -Biocomponent stability studies ; -Analytical validation studies; -Electrode surface characterization with SEM and other microscopic techniques

Partners have excellent computing capabilities possessing clusters with running open source applications totally accessible for researchers working in academics. As for the working environment, institutions offer to the researcher an unrestricted broadband internet access and wider access to many bibliographical resources. The institutions also offers considerably better computing capabilities available also in remote since they possess a computing cluster totally accessible for researchers, with many types of software installed that might be useful for the researcher. This is a great change for researcher working conditions, this computing power offered by host institutions along with the professional assistance of bioinformatics team is difficult available.

4.4 Competences, experience and complementary of the participating organisations and institutional commitment

The project partners are leaders in their respective spheres, having wide experience on nanostructured devices, thus ensuring innovation and a high degree of success in achieving the fundamental science targets of the proposed project.

The UCSD Nanobioelectronic team along with UCM group are already involved in projects for the design of versatile and powerful efficient electrochemical protocols and of sophisticated schemes for their precise analytical control for broad range of practical biomedical and environmental applications. The group has made pioneering contributions towards the design of powerful application of electrochemistry exploring the fundamental aspects of the recognition and transduction events, developing and characterizing new coating materials and electrode transducers, designing new microsensors for clinical diagnostics, environmental monitoring, security surveillance, or industrial process control, exploring flexible wearable (strechable) devices for minimally-invasive on-body sensing (e.g., tattoo-based sensors), developing 'smart' Sense/Act logic-based closed-loop devices, new interfaces for ultrasensitive bioaffinity (DNA, protein) assays, enhancing biodetection through the use of novel materials (e.g., nanowires, nanoparticles) and build compact instruments for field measurement.