Aptamer-based Electrochemical Biosensors for the Detection of Anesthetics

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Aptamers are nucleic-acid sequences capable of recognizing specifically targets of interest. Aptamers are produced via a multi-step artificial evolutionary process, coined 'Systematic Evolution of Ligands by Exponential Enrichment' (SELEX), through which a library (~10^14) of unique sequences is exposed to the target. Competent binders' sequences are recuperated using various means and then amplified using polymer chain reaction. This process is repeated until the desired specificities and affinities for various classes of target (i.e. ions, small molecules, biomolecules and cells) is obtained. Taking advantage of aptamers' versatilities, new biotechnologies have emerged to harness their binding capabilities, but to date none have translated from the laboratory into the 'real-world'. We hypothesize that this poor translation yield is due to: 1) SELEX schemes yield aptamers with good target specificities and affinities but are not optimized for their intended application conditions; 2) SELEX is tedious and implicates several trial-and-error steps leaving the determination of the best viable aptamer challenging; 3) knowledge of the function and structure of aptamers is required to translate them into a successful biotechnology application while few standardized analytical methods have been widely accepted for their characterization; 4) deployment of aptamers into complex matrices has proved to be an important bottleneck where their stabilities and responses fails to address their intended purposes. Taken together, these long-lasting bottlenecks have plagued the promises of aptamers to replace antibodies in biotechnologies. In response, we plan to address these bottlenecks via four objectives. First, we will develop e-SELEX, an original selection scheme tailored to produce aptamers that will be utilized in electrochemical biosensors. As a proof-of-principle for e-SELEX, we aim to develop an aptamer for rocuronium, an aminosteroid neuromuscular blocker commonly employed in the clinic to induce anesthesia in patients in need of an artificial ventilator. Developing e-SELEX is essential to increase aptamers' translation yield into electrochemical biosensors because attachment to surfaces exposes them to electrostatics and entropic-constraints that are currently not considered. Second, we will model aptamers and identify the best candidate from e-SELEX by developing an innovative computational user-friendly approach, E2EDNA, which combines molecular dynamics with machine-learning. Third, we will develop a benchmark analytical method to characterize the aptamer candidates using fluorescence, circular dichroism, nuclear magnetic resonance, isothermal calorimetry and surface plasmon resonance to determine and optimize their functions and structures. Outcomes from this will be used to help refine results from E2EDNA for later selections. Finally, we will translate the aptamer into an electrochemical biosensor that can readily deploy in undiluted whole blood for the detection of rocuronium. Due to the lack of tools available for its therapeutic monitoring, we envision that our electrochemical biosensor will enable seconds-resolved therapeutic monitoring of this rapidly metabolized drug to provide a personalized and on-demand mean of delivery, essential in the context of anesthesia. We envision that our innovative integrative approach from the tailored development of a new receptor down to its application in an electrochemical biosensor, will lead to significant advances for aptamers to improve their translation into new biotechnologies for health monitoring.