Aptamer-based Electrochemical Biosensors for the Detection of Anesthetics

适体是能够识别感兴趣的特异性靶标的核酸序列。适体是通过多步骤人工进化过程产生的，称为“配体的系统进化指数富集”（SELEX），通过该过程，一个库（~10^14）的独特序列暴露于目标。合适的结合物序列通过各种方法得到，然后通过聚合物链反应进行扩增。这一过程反复进行，直到获得各种类型的目标（即离子、小分子、生物分子和细胞）所需的特异性和亲和性。利用适体的多功能性，新的生物技术已经出现，利用它们的结合能力，但到目前为止，还没有一项从实验室转化为“现实世界”。我们假设这种低翻译率是由于：1)SELEX方案产生的适体具有良好的目标特异性和亲和力，但没有针对其预期的应用条件进行优化；2) SELEX是繁琐的，涉及几个试错步骤，使最佳可行适配体的确定具有挑战性；3)将适体转化为成功的生物技术应用需要对其功能和结构的了解，而很少有标准化的分析方法被广泛接受用于其表征；4)将适体部署到复杂矩阵中已被证明是一个重要的瓶颈，因为它们的稳定性和响应未能满足其预期目的。综上所述，这些长期存在的瓶颈一直困扰着适体在生物技术中取代抗体的前景。作为回应，我们计划通过四个目标来解决这些瓶颈。首先，我们将开发e-SELEX，这是一个原创的选择方案，专门用于生产将用于电化学生物传感器的适配体。作为e-SELEX的原理验证，我们的目标是开发罗库溴铵的适配体，罗库溴铵是一种氨基类固醇神经肌肉阻滞剂，通常用于临床需要人工呼吸机的患者诱导麻醉。开发e-SELEX对于提高适体转化为电化学生物传感器的产量至关重要，因为附着在表面会使它们暴露在目前未考虑的静电和熵约束下。其次，我们将通过开发一种创新的计算用户友好方法E2EDNA，将分子动力学与机器学习相结合，对适体进行建模，并从e-SELEX中确定最佳候选者。第三，我们将开发一种基准分析方法，利用荧光、圆二色、核磁共振、等温量热法和表面等离子体共振来表征候选适配体，以确定和优化它们的功能和结构。这一结果将用于帮助改进E2EDNA的结果，以供以后的选择。最后，我们将把适体转化为电化学生物传感器，可以很容易地部署在未稀释的全血中检测罗库溴铵。由于缺乏可用于其治疗监测的工具，我们设想我们的电化学生物传感器将能够对这种快速代谢的药物进行秒分辨率的治疗监测，以提供个性化和按需的递送方式，这在麻醉的背景下是必不可少的。我们设想，我们创新的综合方法，从量身定制的新受体开发到其在电化学生物传感器中的应用，将导致适体的重大进步，以改善其转化为新的生物技术，用于健康监测。

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.