Bioelectronic Sensor using Synthetically Engineered and Electroactive Bacteria for Detection of Aquatic Nutrients

在全球范围内，淡水和海洋生态系统受到多种环境压力的威胁，这些压力包括污染物、入侵物种、气候变化、酸化和营养过剩。生态学家努力监测、理解和模拟过量营养的影响，包括磷酸盐和硝酸盐，以及其他对人类的威胁。理解具有复杂和相互依赖因素的动态空间将需要新一代传感器。生物传感器在灵敏度、容错性、可扩展性、选择性和通用性方面具有显著优势。然而，仅就生物学而言，读数和远程互连目前是有问题的，如果不是不可能的话。生物混合设备可以通过动态生物电子接口来开发和调整电子和生物传感的优势。为了实现这一点，电活性细菌(异化金属/硫酸盐还原细菌)及其胞外电子传递机制被用来传递它们对可测量的生物电流的环境响应。三维纳米软化电极可以收集细菌产生的电流进行信号处理。对环境中的目标的反应更精确地被工程和高度通用的大肠杆菌的集体反应所选择和加强。通过在合成大肠杆菌和异化还原动力希瓦氏杆菌MR-1之间分配传感和驱动角色，通过大量大肠杆菌对电活性细菌群落的个体反应的聚集，增强了对选定靶标存在的信心。生物电子接口设计是迈向新一代传感硬件的基础性一步，这种硬件可以满足机器学习和人工智能的广阔前景。跨学科培训模块将由包括研究生和本科生在内的校园研究人员社区设计。通过纳入新的研究人员，为当地和国际GK-12社区创建了无障碍内容。来自独立媒体独立庇护所自然实验室的当地学生将从肯尼亚基苏木卡拉达混合中学的H20虚拟学院开始，为国际群体制作信息量大、有创意的内容。有天赋的本地和国际学生的国际联系，一系列刺激，传达关于他们周围环境的最新知识，反映了研究的目标。在拟议的工作中，将使用大肠杆菌和S.onedensis MR-1的互联网络来研究检测磷酸盐的机制。磷酸盐检测对于了解水生生态系统中的生态动态是必不可少的。一种动态生物电子接口将被创造出来，用于工程细菌检测多个小分子靶标。该系统的设计本质上是模块化的，其中电活性单胞菌作为生物电子接口，而大肠杆菌是易于工程设计的前端。电活性细菌(异化金属/硫酸盐还原细菌)及其胞外电子传递机制将被用来传递它们对环境的响应，以实现可测量的生物电流。由纳米材料修饰的石墨烯泡沫和这两个细菌组成的三维纳米复合电极将产生并转换用于信号处理的生物电流。对环境中的目标的反应被更精确地选择，并被工程大肠杆菌的集体反应所强化。模块将通过化学群体感应进行连接，并使用合成生物学方法针对传递函数和信号放大进行调整。共培养的细菌配置将在细胞活力和信号传输方面进行优化。这项研究通过以下目标完成：目标1：设计和制造一个从群体感应信号到S.onedensis生物电流的生物电子后端接口，用于信号转导；目标2：设计和制造一个从靶(磷酸盐)到大肠杆菌群体感应信号的生物电子前端接口；目标3：集成目标1和2，并制造、测试和验证从靶(磷)到生物电流信号的生物电子传感器。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Across the globe, freshwater and marine ecosystems are threatened by the effects of multiple, co-occurring environmental pressures including pollutants, invasive species, climate change, acidification, and excess nutrients. Ecologists strive to monitor, understand, and model the effects of excess nutrients, including phosphates and nitrates, in combination with other human threats. Understanding dynamic spaces with complex and interdependent factors will require a new generation of sensors. Biology-enabled sensors have significant advantages with respect to sensitivity, error-tolerance, scalability, selectivity, and versatility. However, readout and long-range interconnectivity are currently problematic, if not impossible, with biology alone. Biohybrid devices can exploit and tune the strengths of both electronic and biological sensing through a dynamic bioelectronic interface. To achieve it, electroactive bacteria (dissimilatory metal/sulfate reducing bacteria) and their extracellular electron transport mechanisms are employed to transduce their environmental response to measurable biocurrent. A three- dimensional nanofabricated electrode can collect a bacterially derived current for signal processing. The response to a target in the environment is more precisely selected and intensified by the collective response of engineered and highly versatile Escherichia coli. By distributing the sensing and actuation roles between synthetic E. coli and the dissimilatory reducing powerhouse Shewanella oneidensis MR-1, confidence in the presence of a select target is enhanced with the aggregation of individual responses from large number of E. coli bacteria to the community of electroactive bacteria. The bioelectronic interface design is a foundational step toward a new generation of sensing hardware that can meet the vast and expanding promise of machine learning and artificial intelligence. Cross-disciplinary training modules will be developed to designed by the on-campus community of researchers including graduate and undergraduate students. By including new researchers, accessible content is created for the local and international GK-12 community. Local students from the Independent Sanctuary for Independent Media Nature Lab will create informative and creative content for the international cohort starting with the H20 Virtual Academy at the Karada Mixed Secondary School in Kisumu, Kenya. The international connection of gifted local and international students, a flow of stimuli, to convey state-of- the-art knowledge about the environment around them reflect the goals of research.In the proposed work, a mechanism for the detection of phosphate will be investigated using an interconnected network of E. coli and S. oneidensis MR-1. Phosphate detection is essential to understanding ecological dynamics in aquatic ecosystems. A dynamic bioelectronic interface will be created for its potential use in the detection of multiple small molecule targets by engineered bacteria. The system design is inherently modular where electroactive S. oneidensis serves as the bioelectronic interface while E.coli is the easily engineered front end. Electroactive bacteria (dissimilatory metal/sulfate reducing bacteria) and their extracellular electron transport mechanisms will be employed to transduce their environmental response to achieve a measurable biocurrent. A three-dimensional nanofabricated electrode, consisting of a nanomaterial-decorated graphene foam and the two bacteria will generate and transduce the biocurrent for signal processing. The response to a target in the environment is more precisely selected and intensified by the collective response of engineered E. coli. Modules will be linked via chemical quorum sensing and tuned with respect to transfer function and signal amplification using synthetic biology approaches. The co-cultured bacteria configuration will be optimized for cell-viability and signal transport. The research is accomplished through the following objectives: Objective 1: Design and fabricate a bioelectronic backend interface from quorum sensing signal to S. oneidensis biocurrent for signal transduction; Objective 2: Design and fabricate a bioelectronic frontend interface from target (phosphate) to E. coli quorum sensing signal; Objective 3: Integrate objectives 1 and 2, and fabricate, test, and validate bioelectronic sensor from target (phosphate) to biocurrent signal.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.