RESULTS
RESULTS OF THE APTASENS PROJECT
Summary of the context and overall objectives of the project
The project was focused on the design of 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.
The conclusions of the action for WP1 were to provide databases with the main physicochemical properties of aptamers binding drugs, VOCs and Flaviviruses. In WP2 it was optimized the immobilization of the aptamers onto transducer surfaces (electrochemical and optical) along with demonstrating the utility of aptamers attached to dynamic and static nanoparticles and delivering the know-how for coupling functionalized aptamers with electrodes surfaces modified with gold nanoparticles. WP 3 tested different electrochemical techniques 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 were considered like piezoelectric transducer suitable for gas sensors detection. WP 4 showed the analytical performances of the aptamers comparing them with classic analytical methods like optical and chromatographic methods. The main conclusions of the action were to produce small density arrays of aptasensors having the sensitivity and reproducibility of the classical analytical methods.
Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far
Three software were used Openeye, Chimera and PyMOL because they provide an academic-free license, 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 was bought with a thiol spacer attached to 5’ 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.
These results can be exploited by selecting specific aptamers for detecting some chemical classes when in presence of other chemical classes.
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 used to produce small density array of aptasensors. Optical and chromatographic methods coupled with tandem mass spectrometry were also used to confirm the performance of the aptamers using routine analytical methods.
The exploitation and dissemination of the work was to deliver experimental procedures more easily, fast and without waste of reagents for the immobilization of aptamers onto electrochemical surfaces.
The main result was to deliver a set of final protocols for the simultaneous detection of multi-analyte using advanced chemometric data processing.
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 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. The results achieved by the project were presented in 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.
Progress beyond the state of the art, expected results until the end of the project and potential impacts
Only few theoretical studies with weak experimental evidence are reported in literature for target-aptamer characterization (virtually and experimentally). The Progress beyond the state of the art obtained by this project was the investigation of target-aptamer interface in terms of geometry, chemical bonds, shape complementarity, affinity constant, thermodynamic and kinetic experimental magnitudes.
This project delivered databases experimentally proved to predict aptasensors performances. In literature, there are NO database available in biosensors area. The project produced different databases of aptamer receptors (also chemically modified) binding ligands in different chemical environments.
The synthesis schemes for increasing signal and stability are well studied with many works increasing every year especially using aptamers. This project released methods to reduce synthesis steps and reagents as much as possible for coupling functionalized aptamers with dynamic or static nanoparticles and electrode surfaces and inks (graphite, silver, gold, platinum).
This project Improved electrochemical portable analytical techniques (sensitivity, selectivity, robustness, drift, stability, reproducibility and long term stability) focusing on miniaturization, wearable, continuous monitoring, in-situ analysis, reagentless, low cost and fast analytical response.
This project implemented small density array of aptasensors for multianalyte detection coupled with multivariate analysis.
The results of the project and potential impacts of them are:
Reinforcing the understanding of the virtual and experimental matching by using large datasets. The Application of advanced chemometric data post-processing to correlate experimental to simulated results was the main improvement achieved by the project.
Producing small density array of rationally designed aptasensors for real samples analysis. This is the major challenge of the analytical chemistry. The impact of this deliverable confirmed the proof of concept of working with rationally designed aptamer and supported the field of virtual assisted bioanalysis.
