A Nicotine Biosensor for Addiction Studies

用于成瘾研究的尼古丁生物传感器

• 批准号: 8647556
• 负责人: Peter A Petillo
• 金额: $ 65.71万
• 依托单位: PINNACLE TECHNOLOGY, INC
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8647556
• 关键词: Achievement; Affect; Animals; Biosensor; Brain; Caffeine; Cessation Research; Cessation of life; Characteristics; Cigarette; Cocaine; Communities; Dependence; Development; Economics; Electronics; Elements; Engineering; Enzymes; Film; Goals; Health Expenditures; Hour; In Vitro; Industry; Kansas; Life; Measures; Membrane; Methods; Microdialysis; Monitor; Mutation; Nicotine; Nicotine Dependence; Oxidases; Performance; Pharmaceutical Preparations; Phase; Positron-Emission Tomography; Process; Productivity; Property; Protein Engineering; Radioimmunoassay; Random Allocation; Reaction Time; Relapse; Research Personnel; Residual state; Rodent; Sales; Scientist; Smoker; Smoking; System; Techniques; Technology; Therapeutic; Time; Tobacco; Tobacco smoking; United States; Universities; Validation; Work; addiction; ascorbate; base; cigarette smoking; cost; design; directed evolution; experience; improved; in vivo; innovation; meetings; neurochemistry; new technology; nicotine oxidase; nicotine replacement; novel; professor; prototype; public health relevance; screening; smoking cessation

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.

摘要 与烟草有关的死亡每年给美国造成约2000亿美元的损失。尼古丁是一种奇特的 对烟草吸烟的依赖形成特性负责，一旦被引入人体， 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现有的研究尼古丁积累的技术成为可能。 在大脑中(例如，微透析、PET、放射免疫分析)不适合在自由活动中进行成瘾研究 动物。生物传感器是一项成熟的技术，可用于监测中枢神经化学物质的实时变化 以一秒一秒为基础的浓度。这项提议的总体目标是交付一个生物传感器 它可以在自由活动的动物体内检测到相关的体内浓度的尼古丁，并且与 顶峰现有的交钥匙系统。生物传感器最关键的组成部分是用作 生物识别元素，生物传感器最终设计的任何方面都不像适当折叠的酶那样重要 足够的活跃度和稳定性。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成部分：(1)6-羟基烟碱氧化酶转化为氧化酶 尼古丁的增强kcat(约0.2秒-1)，同时保持体内KM是预期的1000倍 (2)开发新的更薄的膜来支持尼古丁监测。我们也 成功实施了随机选择和筛选策略，从而产生了新的突变 提高了基因工程尼古丁氧化酶的性能特性。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。我们会 还在继续不断优化新的薄膜，以提高生物传感器的灵敏度。这项工程的完成 该提案将为科学界提供两项重要的创新：1)一种适用于 成瘾研究提供中枢神经系统尼古丁浓度的每秒变化。2)精致的 开发新的生物传感器的方法，这些传感器针对对成瘾和对其没有影响的分析物 目前存在氧化酶(即可卡因和咖啡因)。顶峰将与一个 堪萨斯大学顶尖科学家组成的跨学科联盟。完成后，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，将是第一个 一种用于体内使用的商业化生物传感器，该传感器基于用于分析的工程氧化物酶 以前不存在的酶。