A Nicotine Biosensor for Addiction Studies

烟草相关的死亡每年花费美国大约2000亿美元。尼古丁是吸烟的依赖性形成特性的唯一原因，一旦被引入体内，它会在15秒内影响大脑。打破尼古丁成瘾具有挑战性，复发率仍然很高。尼古丁作用的极快时间尺度使得现有的用于研究尼古丁在大脑中积累的技术（例如，微透析、PET、放射免疫测定）不适合在自由移动的动物中进行成瘾研究。生物传感器是一种经过验证的技术，可用于逐秒监测CNS神经化学物质浓度的实时变化。该提案的总体目标是提供一种生物传感器，该生物传感器可以在相关的体内浓度下检测自由移动的动物体内的尼古丁，并且与品尼高现有的交钥匙系统兼容。生物传感器最关键的组成部分是用作生物识别元件的酶，并且生物传感器的最终设计的任何方面都不如具有足够活性和稳定性特征的正确折叠的酶重要。在第一阶段，我们非常成功地为尼古丁生物传感器的两个关键组件提供了概念验证：（1）将6-羟基尼古丁氧化酶转化为一种对尼古丁具有增强的kcat（~0.2 sec-1）的氧化酶，同时保持Km是预期体内浓度的1000倍，以及（2）开发新的更薄的膜以支持尼古丁监测。我们还成功地实施了随机选择和筛选策略，产生了新的突变，增强了工程尼古丁氧化酶的性能特征。II期将通过尼古丁氧化酶的持续定向进化完成尼古丁酶优化。我们还将继续优化新薄膜，以提高生物传感器的灵敏度。该提案的完成将为科学界提供两项重要创新：1）适用于成瘾研究的尼古丁生物传感器，可提供CNS中尼古丁浓度的逐秒变化。2)一种用于开发新的生物传感器的改进方法，所述生物传感器靶向对成瘾重要的分析物并且目前不存在氧化酶（即，可卡因和咖啡因）。品尼高将与堪萨斯大学的一个由顶尖科学家组成的跨学科联盟合作。完成后，这将是第一个专门设计用于记录啮齿动物中尼古丁的商业生物传感器，并将代表第一个用于体内使用的商业生物传感器，该生物传感器基于以前不存在酶的分析物的工程氧化酶。

DESCRIPTION (provided by applicant): Tobacco-related deaths cost the United States approximately $200 billion each year. Nicotine is singularly responsible for the dependence-forming properties of tobacco smoking and, once introduced to the body, affects the brain within 15 seconds. Breaking nicotine addiction is challenging and relapse rates remain high. The extremely rapid timescale of nicotine action renders existing techniques for studying nicotine accumulation in the brain (e.g., microdialysis, PET, radioimmunoassay) ill-suited for addiction studies in freely moving animals. Biosensors are a proven technology for monitoring real-time changes in CNS neurochemical concentrations on a second-by-second basis. The overall goal of this proposal is the delivery of a biosensor that can detect nicotine in a freely moving animal at relevant in vivo concentrations and that is compatible with Pinnacle's existing turn-key systems. The most critical component of a biosensor is the enzyme used as the biorecognition element, and no aspect of a biosensor's final design is as vital as a properly folded enzyme with sufficient activity and stability profiles. In Phase I, we were highly successful in providing proo-of-concept for two critical components of the nicotine biosensor: (1) conversion of 6-hydroxynicotine oxidase into an oxidase with an enhanced kcat for nicotine (~0.2 sec-1) while maintaining a Km that is 1000-fold above expected in vivo concentrations, and (2) development of new, thinner membranes to support nicotine monitoring. We also successfully implemented a random selection and screening strategy that gave rise to new mutations that enhanced the performance characteristics of the engineered nicotine oxidase enzyme. Phase II will complete the nicotine enzyme optimization through ongoing directed evolution of the nicotine oxidase enzyme. We will also continue the ongoing optimization of new films to improve biosensor sensitivity. The completion of this proposal will provide two important innovations to the scientifc community: 1) A nicotine biosensor suitable for addiction studies that provides second-by-second changes of nicotine concentration in the CNS. 2) A refined approach for the development of new biosensors that target analytes important for addiction and for which no oxidase enzyme currently exists (i.e., cocaine and caffeine). Pinnacle will work in conjunction with an interdisciplinary consortium of leading scientists at the University of Kansas. When completed, this will be the first commercial biosensor specifically designed to record nicotine in rodents and will represent the first commercialized biosensor for in vivo use that is based on an engineered oxidase enzyme for an analyte for which no enzyme previously existed.