A Nicotine Biosensor for Addiction Studies

描述(由申请人提供)：与烟草有关的死亡每年给美国造成约2000亿美元的损失。尼古丁是烟草吸烟形成依赖的特殊原因，一旦进入人体，就会在15秒内影响大脑。戒除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在大脑中积累的技术(如微透析、PET、放射免疫分析)不适合在自由活动的动物中进行成瘾研究。生物传感器是一项成熟的技术，可以每秒监测中枢神经化学物质浓度的实时变化。这项提议的总体目标是提供一种生物传感器，该传感器可以在自由活动的动物体内以相关的体内浓度检测尼古丁，并与Pinnacle现有的交钥匙系统兼容。生物传感器最关键的组件是用作生物识别元件的酶，而生物传感器最终设计的任何方面都不像具有足够活性和稳定性的适当折叠的酶那样重要。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原型：(1)将6-羟基烟碱氧化酶转化为具有增强的尼古丁kcat的氧化酶(~0.2秒-1)，同时保持了比预期体内浓度高1000倍的Km，以及(2)开发了支持尼古丁监测的新的更薄的膜。我们还成功地实施了随机选择和筛选策略，该策略产生了新的突变，从而增强了工程尼古丁氧化酶的性能特征。第二阶段将通过正在进行的尼古丁氧化酶的定向进化来完成尼古丁酶的优化。我们还将继续进行新薄膜的优化，以提高生物传感器的灵敏度。这项提议的完成将为科学界提供两项重要的创新：1)一种适用于成瘾研究的尼古丁生物传感器，它可以提供中枢神经系统中尼古丁浓度的每秒变化。2)一种改进的方法，用于开发新的生物传感器，其目标是对成瘾至关重要的分析物，并且目前不存在对其有氧化酶的分析物(即可卡因和咖啡因)。顶峰公司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。建成后，这将是第一个专门为记录啮齿动物体内尼古丁而设计的商用生物传感器，并将代表着第一个用于活体使用的商业化生物传感器，该传感器基于一种以前没有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.