An Online, Real-Time Microfluidic Biosensor for PFOA and PFOS

获得清洁、可靠的供水对我们的生活质量和我们的经济至关重要，并确保 这种对未来几代人的访问将涉及开发确定安全性的新方法 和饮用水的成分是实用和负担得起的。Per-fl和Per-Poly Uoro烷基物质 全氟辛烷磺酸(PFAS)是困扰美国地下水的最普遍和最持久的污染物之一 国家和人类流行病学研究发现，饮用水中的全氟辛烷磺酸与 一些不利的健康状况，从肝脏和甲状腺疾病到各种形式的癌症。这个 该SBIR方案的主要目标是开发一种可定制的生物传感器平台，该平台使用工程微生物 传感器菌株与Microfluiic技术配对，可持续监测水中的全氟辛烷磺酸。为了达到恶魔的目的- 出于技术可行性的考虑，QBI将执行以下规范fic目标： SPECIfic目标1：鉴定和表征纳米体与全氟辛烷磺酸和全氟辛烷磺酸的结合动力学。 为了使工程菌最大限度地发挥传感器的作用，它必须能够特异地检测fi和 将其目标强烈地捆绑在环境中。QBI将识别结合PFAS分子和 当表面显示在吸附大肠杆菌菌株中时，表征它们的捕获潜力。QBI会奏效的 与当地的纳米体公司abcore合作，分离出一组纳米体，这些纳米体被浓缩用于特定的fi市 然后将这些纳米体带到他们的设施中，将它们克隆到他们的大肠杆菌纳米体中 显示矢量，并在其多路复用的Microfluidic平台中显示和表征它们。 SPECIfic目标2：开发一种用于连续和批量全氟辛烷磺酸传感的原型。为了使用 新开发的传感器菌株在连续监测平台上，QBI将需要开发一种新的AS- 比方说，用于测量来自许多单个菌株库的微米fl标准的凝集度。QBI将从以下方面开始 利用简化的表面展示纳米体集建立和优化凝集试验 菌株，然后他们将在以前的结果的基础上将凝集信号转导到fl-uore- 因此作出回应。最后，QBI将优化一种微型fluiic设备，以促进这种分析并最大限度地提高 细胞fl荧光信号，以便他们可以量化水中存在的污染物的数量。 这些目标的成功完成将有助于验证基于纳米体的传感菌株的使用，以实现 灵敏、选择性和连续的污染物检测，使其对监测旨在 跟踪和评估潜在的危险暴露。无法用来探测许多不同的目标 单一的在线传感器，具有高度的独特性，平台的可定制化和可扩展性 使用合成生物学来改造菌株是一种变革性的方法。这将使QBI不断扩大 他们的客户群，因为他们不断增加为满足最终用户需求而量身定做的传感功能。 1

Access to clean, reliable water supplies is critical to our quality of life and our economy, and ensuring this access for generations to come will involve developing novel approaches to determining the safety and composition of potable water that are practical and affordable. Per- and polyfluoroalkyl substances (PFASs) are among the most ubiquitous and persistent contaminants plaguing groundwater in the United States, and human epidemiological studies have found associations between PFASs in drinking water and a number of adverse health conditions, from liver and thyroid disorders to various forms of cancer. The main objective of this SBIR proposal is to develop a customizable biosensor platform that uses engineered microbial sensor strains paired with microfluidic technology to continuously monitor water for PFASs. In order to demon- strate technical feasibility, QBI will perform these Specific Aims: Specific Aim 1: To identify and characterize the binding kinetics of nanobodies for PFOA and PFOS. For an engineered bacterial strain to be maximally effective as a sensor, it must be able to specifically and strongly bind its target in the environment. QBI will identify nanobodies that bind PFAS molecules and characterize their capture potential when surface displayed in an adsorbing E. coli strain. QBI will work with a local nanobody company, Abcore, to isolate a set of nanobodies that are enriched for specificity to the two targets and will then bring these nanobodies to their facility, clone them into their E. coli nanobody display vector, and screen and characterize them within their multiplexed microfluidic platform. Specific Aim 2: To develop a prototype for continuous and batch PFAS sensing. In order to use the newly developed sensor strains in a continuous monitoring platform, QBI will need to develop a novel as- say for measuring agglutination on a microfluidic-scale from many individual strain banks. QBI will begin by developing and optimizing an agglutination assay using the reduced set of surface-displayed nanobody strains, and then they will build upon previous results to transduce the agglutination signal to a fluores- cence response. Finally, QBI will optimize a microfluidic device to facilitate this assay and maximize the cellular fluorescence signal so that they can quantify the amount of contaminant present in the water. Successful completion of these Aims will serve to validate the use of nanobody-based sensing strains to achieve sensitive, selective, and continuous contaminant detection, making it of great utility to monitoring efforts aimed at tracking and assessing potential hazardous exposures. Beyond the ability to detect many different targets with a single on-line sensor, which is highly unique, the customizability and expandability of the platform using synthetic biology to engineer strains is transformative. This will enable QBI to continually expand their customer base as they continue to add sensing capabilities tailored to meet end-users' needs. 1