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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - APTASENS (A new generation of AptaSensors)

Teaser

Summary

The project was focused on the design different next generation libraries of biosensors that will yield ready-to-go analytical tools against any target in unprecedented speed and high affinity. Aptamers (DNA and peptides), a new generation of recognition elements were computationally selected and integrated into bioelectronics devices.
The huge number of candidates to be tested using a combinatorial trial and error approach was radically reduced by rationalizing the strategy to design the aptamers. New virtual screening processes were developed for the generation of aptamers by the help of modern molecular modelling tools.
Databases with the main physicochemical properties of aptamers binding drugs, VOCs and Flaviviruses were produced. These results can be exploited by selecting specific aptamers for detecting some chemical classes when in presence of other chemical classes.
The project also explored the chemistry of the aptamers for the Immobilization and stability onto the electrochemical transducer of the electronic device. The performances of the aptamers attached to dynamic and static nanoparticles, especially gold nanoparticles were experimentally studied. The Know-how for coupling functionalized aptamers with graphite electrode surfaces modified with Gold nanoparticles electrodeposited were also provided. The main exploitation of this part was to deliver experimental procedures more easily, fast and without waste of reagents for the immobilization of aptamers onto electrochemical surfaces.
Moreover during this period, different electrochemical techniques was studied especially to have a label-free detection. Electrochemical impedance spectroscopy was widely studied because of the its unique property to explore the electrochemical surface without any label. Other electrochemical devices was considered like piezoelectric transducer suitable for gas sensors detection. The analytical performances of the aptamers were also proved by classic analytical methods like optical and chromatographic methods coupled with tandem mass spectrometry.

Work performed

Two software were selected Openeye and Chimera because they provide an academic-free licence they have good flexibility of customization and help support. The customization of the in silico procedure was done using python and Bash scripts allowing automation of all algorithm steps.
Two kind of libraries were designed. The first was based on the ssDNA designed only using the four natural bases adenine (A), cytosine (C), guanine (G) and thymine (T). The second was based on aminoacid motif. Because of the huge combinations the docking process was run in different steps. In each step a peptide library was generated by using an incremental construction approach. In every subsequent iteration, a focused library of peptides of increasing complexity, was built on previous iteration results.
Aptamers were chemically modified using thiol-Au covalent binding, EDC/NHS chemistry and biotin link in various synthesis format. The ssDNA were bought with a thiol spacer attached to 5â€™ end of the DNA to be quickly bound to gold nanoparticles (AuNPs). In sensors, the use of AuNP as platform for target binding was found to increase the sensitivity by two orders of magnitude versus monolayer modified Au surface.
Two instruments quartz crystal microbalance (QCM) and miniaturized potentiostat with screen printed electrodes for rapid (seconds or minutes) determination of targets, in the range of ppm to ppb, were tested. Optical and chromatographic methods coupled with tandem mass spectrometry were also used to confirm the performance of the aptamers using routine analytical methods.
The research training objectives were achieved following periodically seminars and/or workshops on the resolution of specific tasks related to the project acquiring additional scientific and complementary skills. The researcher was supported and supervised for step by step working day by scientists in charge and experienced researchers team of the outgoing and also in remote by the return institution. A dedicated website was created for remote assistance, sharing and comments along with the use of social media like research-gate.
The researcher participated actively in different international conferences. In particular, the data obtained in the project were presented: In the conference NanoFlorida The 10th Annual Symposium took in 2017 where the researcher was invited to do a talk and he presented a poster; In 2018, the researcher gave two talks (one invited) in the Biosensors 2018 the most followed conference in the world on sensors and biosensors. During the period covered by this report, three publications on scientific international journals with referees were published. Three other publications are in progress. The researcher was in all publications corresponding author highlighting his capacity in reaching and re-enforcing the professional maturity in research.

Final results

Up to now, the Characterization (virtually and experimentally) of the interaction of target-aptamer including chemical environment phenomena is not explored and there are only few theoretical studies with weak experimental evidence. The Progress beyond the state of the art obtained by this project was the analysis of ligand-receptor interface in terms of geometry, chemical bonds, shape complementarity, affinity constant, compared with thermodynamic and kinetic experimental magnitudes.
Moreover in this project, an important deliverable was to produce databases experimentally proved to predict aptasensors performances. In literature, there are NO database available especially in biosensors area. The project produced different databases of aptamer receptors (eventually chemically modified) binding ligands in different chemical environments.
In experimental part, the synthesis schemes for increasing signal and stability are well studied there many works increasing every year especially using aptamers. In this way the project contributed to propose methods to reduce synthesis steps and reagents as much as possible for coupling functionalized aptamers with electrode surfaces and inks (graphite, silver, gold, platinum).
The project Improved electrochemical portable analytical techniques (sensitivity, selectivity, robustness, drift, stability, reproducibility and long term stability). This point is huge explored by many laboratories world-wide but there are still few application in aptasensors. The project was focused on opening new frontiers in miniaturization, wearable, continuous monitoring, in-situ analysis, reagentless, low cost and fast analytical response.
The expected results until the end of the project and potential impacts of them are:
Applying the technology released in this period to analyze real samples by aptasensors. This is the major challenge of the analytical chemistry, but often leads to the realization of â€œacademicâ€ demonstration and very few applications in real samples are reported. The impact of this objective will be to bridge the gap of major drawbacks in biosensor area and confirm the proof of concept.
Modelling analysis by considerable datasets. Now, there are insufficient datasets on aptasensors especially supported by simulation. The Application of advanced chemometric data post-processing to correlate experimental results to simulated one is the main improvement that will be achieved by the project in the future.