A Nicotine Biosensor for Addiction Studies

摘要 与烟草有关的死亡每年使美国损失约2000亿美元。尼古丁是一种 负责烟草吸烟的依赖性形成特性，一旦被引入体内， 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研究尼古丁积累的技术 在大脑中（例如，微透析，PET，放射免疫测定）不适合在自由移动的成瘾研究 动物生物传感器是一种成熟的技术，用于监测中枢神经系统神经化学物质的实时变化， 浓度在每秒的基础上。该提案的总体目标是提供生物传感器 其可以在相关的体内浓度下检测自由移动的动物中的尼古丁， Pinnacle现有的交钥匙系统。生物传感器最关键的组成部分是用作生物传感器的酶。 生物识别元件，生物传感器的最终设计的任何方面都不如正确折叠的酶重要， 足够的活性和稳定性。在第一阶段，我们非常成功地提供了概念验证， 尼古丁生物传感器的两个关键组件：（1）将6-羟基尼古丁氧化酶转化为氧化酶 尼古丁的kcat增强（约0.2 sec-1），同时保持Km高于体内预期1000倍 浓度，和（2）开发新的，更薄的膜，以支持尼古丁监测。我们也 成功地实施了随机选择和筛选策略，产生了新的突变， 增强了工程化尼古丁氧化酶的性能特征。第二阶段将完成 通过尼古丁氧化酶的持续定向进化来优化尼古丁酶。我们将 我们还将继续优化新薄膜，以提高生物传感器的灵敏度。完成这一 该提案将为科学界提供两个重要的创新：1）尼古丁生物传感器适用于 成瘾研究提供了中枢神经系统中尼古丁浓度的逐秒变化。2)一个精致 开发新的生物传感器的方法，该生物传感器靶向对成瘾重要的分析物， 目前存在氧化酶（即，可卡因和咖啡因）。Pinnacle将与 由堪萨斯大学的顶尖科学家组成的跨学科联盟。完成后，这将是 第一个专门设计用于记录啮齿动物体内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化生物传感器，其基于用于分析物的工程化氧化酶， 以前没有酶存在。

ABSTRACT 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 proof-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 scientific 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.