Aptamer-based biosensors as a new analytical tool for neuroscience

Electrochemical sensors have the capability to sensitively and reliably detect target molecules in complex media. Aptamers are single stranded nucleotide sequences capable of specifically and selectively bind a large variety of target molecules. The purpose of this project, comprising three main parts, was to establish an aptamer based electrochemical platform as a new analytical method for the real-time continuous monitoring of small molecules released in the brain. In the first part, the protocol for the production of an aptamer-based electrochemical sensing platform capable of continuous monitoring was established and optimised using the ATP/adenosine aptamer as a model. The redox-decorated aptamer was immobilised on a gold surface and the signal generated by the displacement of the electron donor upon binding of the target molecule with the DNA strand. The fabrication and its properties were assessed via cyclic voltammetry (CV) and the sensor response recorded via square wave voltammetry (SWV). Three aptamers for ATP/ Adenosine were employed and their performance on the platform was evaluated both against the target and the downstream molecules. In the second part, the characterised platform was employed to record a biological event: the in vitro release of adenosine from a hypoxic brain slice. Prior to performing the measurement that required tissue, all the control measurements were performed to evaluate influence of different environmental factors e.g. pH, temperature, buffer composition. Another preliminary measurement aimed to verify whether the sensor could be inserted in the tissue, by recording the sensor response whilst inserted in an agar block. Lastly the recording of adenosine release from hypoxic brain tissue was performed simultaneously with electrophysiology recordings. In the third and last part capture-SELEX was employed to select two novel structure-switching aptamers against UTP and UDP-glucose, two neurochemicals connected to pathological conditions such as epilepsy, dry eye disease and metastatic cancer. The sequenced libraries were then analysed and the emerged candidates were at first screened with SYBRGreen assay, a high throughput fluorescence assay, and the results confirmed via microscale thermophoresis (MST). The final selected aptamer, for each target molecule, underwent the first truncation round. Finally, the binding affinity of the two obtained sequences were characterised via MST