Periodic Reporting for period 1 - eADAM (Ultra-small electrochemical aptasensors for specific dopamine real-time monitoring) Période du rapport: 2016-02-01 au 2018-01-31 Résumé du contexte et des objectifs généraux du projet Psychiatric and neurodegenerative diseases (ND) associated with malfunctions in the brain´s neurotransmitter metabolism put a burden on the society shoulders. Only the costs of dementia approach 1% of world's GDP (US $818 billion in 2015) from where approximately 40% are derived from non-medical services (e.g. social services, special accommodation or informal care). Even more shocking is that a large majority of people living with ND do not receive a formal diagnosis, meaning they do not have access to treatment, care and/or support. A large number of these disorders (e.g. Parkinson’s and Alzheimer’s diseases, schizophrenia, drug addiction, or psychosis) are correlated with abnormal fluctuations in the levels of dopamine (a small molecule neurotransmitter secreted by the neurons in the brain). Therefore, it is not surprising that dopamine occupies a central piece in the current neuroscience research, aiming to understand the mechanism of the disease development and early diagnosis.The intrinsic electroactive nature of dopamine makes electrochemical methods very well suited for its monitoring. However, other co-existing molecules, such as nor/epinephrine, L-DOPA, catechol or ascorbic acid, provide signals in the range of potentials where dopamine is oxidized, thereby questioning the chemical selectivity of the measurements. The overall objective of this project was to develop electrochemical tools that ensure the specific measure of dopamine by this contributing to redefine the way dopaminergic diseases are monitored and treated. Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent This project started showing that the RNA aptamer sequence specifically designed to recognize dopamine could be immobilized on the gold electrode surface keeping its selectivity. The approach consisted in employing a C6 alkanethiol linker at the 5’ end of the sequence and a C2 amine terminated alkanethiol as blocking agent. Thanks to the careful design, the aptasensor allowed discrimination between dopamine and interfering species, including related catecholamines (L-DOPA, catechol and nor/epinephrine) and ascorbic acid. With this in hand, we designed and developed a flow-injection wall-jet analytical platform for the aptasensor to be coupled. The analytical system allowed detection of dopamine in small volumes of untreated human biological samples at its physiological relevant range (0.01—1 µM) and with a response time of less than 1s.We also demonstrated that the replacement of the RNA bases by their DNA analogues destroys the aptamer specificity of biorecognition and binding of dopamine, providing the same response as a DNA mutated sequence with destabilized dopamine-binding region.We explored the possibility of using redox-labelled dendrimers as wires to improve sensitivity. This resulted to be a particularly interesting system, where the electron transfer (ET) mechanism was strongly influenced by the nanostructure arrangement at the electrode surface. In the case of diluted layers, the ET followed the features of a surface-confined reaction that occurs by electron tunneling, while in compact monolayers the mechanism of ET formally switched to a diffusion-controlled process. This was attributed to the strong interactions existing between the neighboring dendrimers, which restricted their motional freedom and affected their conformational state on the electrode surface.The next step in the project was to adapt the specific dopamine sensor for implantation in Drosophila flies and for that, carbon materials resulted to be more adequate. We found that the electrochemical reactions of dopamine and related catecholamines were slowed down both kinetically and thermodynamically at electrode materials with graphite-layered structure. Interestingly, the reactions proceeded at different potentials, so by using amperometry it was possible to discriminate dopamine from interferents in both cerebrospinal and hemolymph-like fluids. The microelectrodes were finally used for implantation and dopamine recordings in Drosophila flies.The overall scientific outcome from the project was deposited in the open access repository PURE and disseminated to:- The scientific community through 5 high-impact factor peer reviewed publications and 4 oral presentations at international conferences.- The public through volunteer lectures. Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent) The results from this project put our collaborators in a unique position to advance in the knowledge of one of the priority areas of our society: neurodegenerative diseases, by improving the understanding of dopamine neuron systems and correlating fluctuations with external factors in real-time. Moreover, it has stablished a network of scientists with complementary expertise that will continue to drive advances in the field of biomedical research. fig-for-report.jpg
