Aptamer-based Electrochemical Biosensors for the Detection of Anesthetics

用于检测麻醉剂的基于适体的电化学生物传感器

• 批准号: 571442-2021
• 负责人: DauphinDucharme, PhilippeP
• 金额: $ 3.28万
• 依托单位: Université de Sherbrooke
• 依托单位国家: 加拿大
• 项目类别: Alliance Grants
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=748591
• 关键词: Aptamer; based; Electrochemical; Biosensors; Detection

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.

适体是能够识别特定目标靶标的核酸序列。适体是通过多步人工进化过程产生的，即通过指数富集对配体的系统演变”（SELEX），通过该过程，通过该过程，唯一序列的库（〜10^14）通过该过程暴露于目标。使用各种手段对胜任的粘合剂序列进行恢复，然后使用聚合物链反应进行扩增。重复此过程，直到获得各种靶标的所需的特异性和亲和力（即离子，小分子，生物分子和细胞）。利用适体的多功能性，已经出现了新的生物技术来利用其具有约束力的能力，但迄今为止，没有一个从实验室转化为“现实世界”。我们假设这种不良的翻译产量是由于：1）SELEX方案产生的适体具有良好的目标特异性和亲和力，但并未针对其预期的应用条件进行优化； 2）SELEX很乏味，暗示了几个反复试验的步骤，这使得确定最佳适应性挑战。 3）需要适体功能和结构的知识来将其转化为成功的生物技术应用，而很少有标准化的分析方法被广泛接受以进行表征； 4）事实证明，将适体部署到复杂的矩阵中是一个重要的瓶颈，其稳定性和响应无法解决其预期目的。综上所述，这些持久的瓶颈困扰着适体代替生物技术中的抗体的承诺。作为回应，我们计划通过四个目标解决这些瓶颈。首先，我们将开发E-SELEX，这是一种原始的选择方案，该方案量身定制，用于生产适合于电化学生物传感器的适体。作为E-SELEX的原则证明，我们旨在为诊所通常使用的氨基固醇肌肉肌肉阻滞剂开发适体，以诱导需要人造呼吸机的患者进行麻醉。开发E-核心对于增加适体的翻译产量至电化学生物传感器至关重要，因为对表面的依恋使它们暴露于目前未考虑的静电和熵构造物中。其次，我们将通过开发创新的计算用户友好方法E2EDNA来对适体进行建模，并确定E-SELEX的最佳候选者，该方法将分子动力学与机器学习结合在一起。第三，我们将开发一种基准分析方法，以使用荧光，圆形二色性，核磁共振，等温量热法和表面等离子体共振来表征适体候选物，以确定和优化其功能和结构。此结果将用于帮助优化E2EDNA的结果以进行以后的选择。最后，我们将把适体转换为电化学生物传感器，可以轻易部署未稀释的全血以检测Rocuronium。由于缺乏用于治疗监测的工具，我们设想我们的电化学生物传感器将对这种快速代谢的药物进行秒分解的治疗监测，以提供个性化和按需的递送平均值，对麻醉的背景至关重要。我们设想，从量身定制的新受体开发到在电化学生物传感器中的应用中，我们的创新综合方法将导致适体将其转化为新的生物技术进行健康监测的新生物技术。